Dig-3 insecticidal cry toxins

ABSTRACT

DIG-3 Cry toxins, polynucleotides encoding such toxins, and transgenic plants that produce such toxins are useful to control insect pests.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims benefit to U.S. Provisional Patent ApplicationNo. 61/170,189, filed Apr. 17, 2009, which is expressly incorporated byreference herein.

FIELD OF THE INVENTION

This invention concerns new insecticidal Cry toxins and their use tocontrol insects.

BACKGROUND OF THE INVENTION

Bacillus thuringiensis (B.t.) is a soil-borne bacterium that producespesticidal crystal proteins known as delta endotoxins or Cry proteins.Cry proteins are oral intoxicants that function by acting on midgutcells of susceptible insects. An extensive list of delta endotoxins ismaintained and regularly updated athttp://www.lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/intro.html.

European corn borer (ECB), Ostrinia nubilalis (Hübner), is the mostdamaging insect pest of corn throughout the United States and Canada,and causes an estimated $1 billion revenue loss each year due to cropyield loss and expenditures for insect management (Witkowski et al.,2002). Transgenic corn expressing genes encoding Cry proteins, mostnotably Cry1Ab, Cry1Ac, or Cry1F, provide commercial levels of efficacyagainst ECB.

Despite the success of ECB-resistant transgenic corn, the possibility ofthe development of resistant insect populations threatens the long-termdurability of Cry proteins in ECB control and creates the need todiscover and develop new Cry proteins to control ECB and other pests.Insect resistance to B.t. Cry proteins can develop through severalmechanisms (Heckel et al., 2007, Pigott and Ellar, 2007). Multiplereceptor protein classes for Cry proteins have been identified withininsects, and multiple examples exist within each receptor class.Resistance to a particular Cry protein may develop, for example, bymeans of a mutation within the toxin-binding portion of a cadherindomain of a receptor protein. A further means of resistance may bemediated through a protoxin-processing protease. Thus, resistance to Crytoxins in species of Lepidoptera has a complex genetic basis, with atleast four distinct, major resistance genes. Lepidopteran insectsresistant to Cry proteins have developed in the field within the speciesPlutella xylostella (Tabashnik, 1994), Trichoplusia ni (Janmaat andMyers 2003, 2005), and Helicoverpa zeae (Tabashnik et al., 2008).Development of new high potency Cry proteins would provide additionaltools for management of ECB and other insect pests. Cry proteins withdifferent modes of action produced in combination in transgenic cornwould prevent the development ECB insect resistance and protect the longterm utility of B.t. technology for insect pest control.

BRIEF SUMMARY OF THE INVENTION

The present invention provides insecticidal Cry toxins, including thetoxin designated herein as DIG-3 as well as variants of DIG-3, nucleicacids encoding these toxins, methods of controlling pests using thetoxins, methods of producing the toxins in transgenic host cells, andtransgenic plants that produce the toxins. The predicted amino acidsequence of the wild type DIG-3 toxin is given in SEQ ID NO:2.

As described in Example 1, a nucleic acid encoding the DIG-3 protein wasisolated from a B.t. strain internally designated by Dow AgroSciencesLLC as PS46L. The nucleic acid sequence for the full length codingregion was determined, and the full length protein sequence was deducedfrom the nucleic acid sequence. The DIG-3 toxin has some similarity toCry1BII (Genbank Accession No. AAM93496) and other B. thuringiensisCry1B-type proteins(http://www.lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/intro.html).

Insecticidally active variants of the DIG-3 toxin are also describedherein, and are referred to collectively as DIG-3 toxins.

DIG-3 toxins may also be used in combination with RNAi methodologies forcontrol of other insect pests. For example, DIG-3 can be used intransgenic plants in combination with a dsRNA for suppression of anessential gene in corn rootworm or an essential gene in an insect pest.Such target genes include, for example, vacuolar ATPase, ARF-1, Act42A,CHD3, EF-1α, and TFIIB. An example of a suitable target gene is vacuolarATPase, as disclosed in WO2007/035650.

A surprising finding reported herein is that DIG-3 toxins are activeagainst populations of European corn borer and diamond back moth thatare resistant to Cry1F and Cry1A toxins. Accordingly, DIG-3 toxins areideal candidates for use to control of Lepidopteran pests. The toxinscan be used alone or in combination with other Cry toxins, such asCry1F, Cry1Ab, and Cry1Ac, to control development of resistant insectpopulations.

Insecticidally active fragments of SEQ ID NO:2, and nucleotides encodingsuch fragments, are another aspect of the invention.

In one embodiment the invention provides an isolated DIG-3 toxinpolypeptide comprising a core toxin segment selected from the groupconsisting of

-   -   (a) a polypeptide comprising the amino acid sequence of residues        113 to 643 of SEQ ID NO:2;    -   (b) a polypeptide comprising an amino acid sequence having at        least 90% sequence identity to the amino acid sequence of        residues 113 to 643 of SEQ ID NO:2;    -   (c) a polypeptide comprising an amino acid sequence of residues        113 to 643 of SEQ ID NO:2 with up to 20 amino acid        substitutions, deletions, or modifications that do not adversely        affect expression or activity of the toxin encoded by SEQ ID        NO:2.

In one embodiment the invention provides an isolated DIG-3 toxinpolypeptide comprising a core toxin segment selected from the groupconsisting of

-   -   (a) a polypeptide comprising the amino acid sequence of residues        73 to 643 of SEQ ID NO:2;    -   (b) a polypeptide comprising an amino acid sequence having at        least 90% sequence identity to the amino acid sequence of        residues 73 to 643 of SEQ ID NO:2;    -   (c) a polypeptide comprising an amino acid sequence of residues        73 to 643 of SEQ ID NO:2 with up to 20 amino acid substitutions,        deletions, or modifications that do not adversely affect        expression or activity of the toxin encoded by SEQ ID NO:2.

In another embodiment the invention provides an isolated DIG-3 toxinpolypeptide comprising a DIG-3 core toxin segment selected from thegroup consisting of

-   -   (a) a polypeptide comprising the amino acid sequence of residues        1 to 643 of SEQ ID NO:2;    -   (b) a polypeptide comprising an amino acid sequence having at        least 90% sequence identity to the amino acid sequence of        residues 1 to 643 of SEQ ID NO:2;    -   (c) a polypeptide comprising an amino acid sequence of residues        1 to 643 of SEQ ID NO:2 with up to 20 amino acid substitutions,        deletions, or modifications that do not adversely affect        expression or activity of the toxin encoded by SEQ ID NO:2.

By “isolated” applicants mean that the polypeptide or DNA molecules havebeen removed from their native environment and have been placed in adifferent environment by the hand of man.

In another embodiment the invention provides a plant comprising a DIG-3toxin.

In another embodiment the invention provides a method for controlling apest population comprising contacting said population with apesticidally effective amount of a DIG-3 toxin.

In another embodiment the invention provides an isolated nucleic acidthat encodes a DIG-3 toxin.

In another embodiment the invention provides a DNA construct comprisinga nucleotide sequence that encodes a DIG-3 toxin operably linked to apromoter that is not derived from Bacillus thuringiensis and is capableof driving expression in a plant. The invention also provides atransgenic plant that comprises the DNA construct stably incorporatedinto its genome and a method for protecting a plant from a pestcomprising introducing the construct into said plant.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 DNA sequence encoding full-length DIG-3 toxin; 3771 nt.

SEQ ID NO:2 Full-length DIG-3 protein sequence; 1256 aa.

SEQ ID NO:3 Plant-optimized full length DIG-3 DNA sequence; 3771 nt.

SEQ ID NO:4 Cry1Ab protoxin segment; 545 aa.

SEQ ID NO:5 Chimeric toxin: DIG-3 Core toxin segment/Cry1Ab protoxinsegment; 1188 aa.

SEQ ID NO:6 Dicot-optimized DNA sequence encoding the Cry1Ab protoxinsegment; 1635 nt

SEQ ID NO:7 Maize-optimized DNA sequence encoding the Cry1Ab protoxinsegment; 1635 nt

DETAILED DESCRIPTION OF THE INVENTION

DIG-3 Toxins, and insecticidally active variants. In addition to thefull length DIG-3 toxin of SEQ ID NO:2, the invention encompassesinsecticidally active variants. By the term “variant”, applicants intendto include fragments, certain deletion and insertion mutants, andcertain fusion proteins. DIG-3 is a classic three-domain Cry toxin. As apreface to describing variants of the DIG-3 toxin that are included inthe invention, it will be useful to briefly review the architecture ofthree-domain Cry toxins in general and of the DIG-3 protein toxin inparticular.

A majority of Bacillus thuringiensis delta-endotoxin crystal proteinmolecules are composed of two functional segments. Theprotease-resistant core toxin is the first segment and corresponds toabout the first half of the protein molecule. The full ˜130 kDa protoxinmolecule is rapidly processed to the resistant core segment by proteasesin the insect gut. The segment that is deleted by this processing willbe referred to herein as the “protoxin segment”. The protoxin segment isbelieved to participate in toxin crystal formation (Arvidson et al.,1989). The protoxin segment may thus convey a partial insect specificityfor the toxin by limiting the accessibility of the core to the insect byreducing the protease processing of the toxin molecule (Haider et al.,1986) or by reducing toxin solubility (Aronson et al., 1991). B.t.toxins, even within a certain class, vary to some extent in length andin the precise location of the transition from the core toxin segment toprotoxin segment. The transition from core toxin segment to protoxinsegment will typically occur at between about 50% to about 60% of thefull length toxin. SEQ ID NO:2 discloses the 1256 amino acid sequence ofthe full-length DIG-3 polypeptide, of which the N-terminal 643 aminoacids comprise the DIG-3 core toxin segment. The 5′-terminal 1929nucleotides of SEQ ID NO:1 comprise the coding region for the core toxinsegment.

Three dimensional crystal structures have been determined for Cry1Aa1,Cry2Aa1, Cry3Aa1, Cry3Bb1, Cry4Aa, Cry4Ba and Cry8Ea1. These structuresfor the core toxins are remarkably similar and are comprised of threedistinct domains with the features described below (reviewed in de Maagdet al., 2003).

Domain I is a bundle of seven alpha helices where α-helix 5 issurrounded by six amphipathic helices. This domain has been implicatedin pore formation and shares homology with other pore forming proteinsincluding hemolysins and colicins. Domain I of the DIG-3 proteincomprises amino acid residues 56 to 278 of SEQ ID NO:2.

Domain II is formed by three anti-parallel beta sheets packed togetherin a beta prism. The loops of this domain play important roles inbinding insect midgut receptors. In Cry1A proteins, surface exposedloops at the apices of Domain II beta sheets are involved in binding toLepidopteran cadherin receptors. Cry3Aa Domain II loops bind amembrane-associated metalloprotease of Leptinotarsa decemlineata (Say)(Colorado potato beetle) in a similar fashion (Ochoa-Campuzano et al.,2007). Domain II shares homology with certain carbohydrate-bindingproteins including vitelline and jacaline. Domain II of the DIG-3protein comprises amino acid residues 283 to 493 of SEQ ID NO:2.

Domain III is a beta sandwich of two anti-parallel beta sheets.Structurally this domain is related to carbohydrate-binding domains ofproteins such as glucanases, galactose oxidase, sialidase and others.Domain III binds certain classes of receptor proteins and perhapsparticipates in insertion of an oligomeric toxin pre-pore that interactswith a second class of receptors, examples of which are aminopeptidaseand alkaline phosphatase in the case of Cry1A proteins (Pigott andEllar, 2007). Analogous Cry Domain III receptors have yet to beidentified in Coleoptera. Conserved B.t. sequence blocks 2 and 3 mapnear the N-terminus and C-terminus of Domain 2, respectively. Hence,these conserved sequence blocks 2 and 3 are approximate boundary regionsbetween the three functional domains. These regions of conserved DNA andprotein homology have been exploited for engineering recombinant B.t.toxins (U.S. Pat. No. 6,090,931, WO 91/01087, WO 95/06730, WO1998022595). Domain III of the DIG-3 protein comprises amino acidresidues 503 to 641 of SEQ ID NO:2.

It has been reported that α-helix 1 of Domain I is removed followingreceptor binding. Aronson et al. (1999) demonstrated that Cry1Ac boundto BBMV was protected from proteinase K cleavage beginning at residue59, just after α-helix 1; similar results were cited for Cry1Ab. Gomezet al. (2002) found that Cry1Ab oligomers formed upon BBMV receptorbinding lacked the α-helix 1 portion of Domain I. Also, Soberon et al.(2007) have shown that N-terminal deletion mutants of Cry1Ab and Cry1Acwhich lack approximately 60 amino acids encompassing α-helix 1 on thethree dimensional Cry structure are capable of assembling monomers ofmolecular weight about 60 kDa into pre-pores in the absence of cadherinbinding. These N-terminal deletion mutants were reported to be active onCry-resistant insect larvae. Furthermore, Diaz-Mendoza et al. (2007)described Cry1Ab fragments of 43 kDa and 46 kDa that retained activityon Mediterranean corn borer (Sesamia nonagrioides). These fragments weredemonstrated to include amino acid residues 116 to 423; however theprecise amino acid sequences were not elucidated and the mechanism ofactivity of these proteolytic fragments is unknown. The results of Gomezet al. (2002), Soberon et al. (2007), and Diaz-Mendoza et al. (2007)contrast with those of Hofte et al. (1986), who reported that deletionof 36 amino acids from the N-terminus of Cry1Ab resulted in loss ofinsecticidal activity.

We have deduced the beginnings and ends of α-helix 1, α-helix 2A,α-helix 2B, and α-helix 3, and the location of the spacer regionsbetween them in Domain I of the DIG-3 toxin by comparing the DIG-3protein sequence with the protein sequence for Cry8Ea1, for which thestructure is known. These locations are described in Table 1.

TABLE 1 Amino acid coordinates of projected α-helices of DIG-3 protein.α-helix 1 spacer α-helix 2A spacer α-helix 2B spacer α-helix 3 Residuesof 53-70 71-76 77-91 92-99 100-108 109-113 114-138 SEQ ID NO: 2

Amino terminal deletion variants of DIG-3. In one of its aspects theinvention provides DIG-3 variants in which all or part of α-helix 1,α-helix 2A, and α-helix 2B are deleted to improve insecticidal activityand avoid development of resistance by insects. These modifications aremade to provide DIG-3 variants with improved attributes, such asimproved target pest spectrum, potency, and insect resistancemanagement. In some embodiments of the invention, the subjectmodifications may affect the efficiency of protoxin activation and poreformation, leading to insect intoxication. More specifically, to provideDIG-3 variants with improved attributes, step-wise deletions aredescribed that remove part of the nucleic acid sequence encoding theN-terminus of the DIG-3 protein. The deletions remove all of α-helix 1and all or part of α-helix 2 in Domain I, while maintaining thestructural integrity of α-helices 3 through 7. The subject inventiontherefore relates in part to improvements to Cry protein efficacy madeby engineering the α-helical components of Domain 1 for more efficientpore formation. More specifically, the subject invention relates in partto improved DIG-3 proteins designed to have N-terminal deletions inregions with putative secondary structure homology to α-helix 1 andα-helix 2 in Domain I of Cry1 proteins.

Deletions to improve the insecticidal properties of the DIG-3 toxins mayinitiate before the predicted α-helix 2A start, and may terminate afterthe α-helix 2B end, but preferably do not extend into α-helix 3.

In designing coding sequences for the N-terminal deletion variants, anATG start codon, encoding methionine, is inserted at the 5′ end of thenucleotide sequence designed to encode the deletion variant. Forsequences designed for use in transgenic plants, it may be of benefit toadhere to the “N-end rule” of Varshaysky (1997). It is taught that someamino acids may contribute to protein instability and degradation ineukaryotic cells when displayed as the N-terminal residue of a protein.For example, data collected from observations in yeast and mammaliancells indicate that the N-terminal destabilizing amino acids are F, L,W, Y, R, K, H, I, N, Q, D, E and possibly P. While the specifics ofprotein degradation mechanisms may differ somewhat between organisms,the conservation of identity of N-terminal destabilizing amino acidsseen above suggests that similar mechanisms may function in plant cells.For instance, Worley et al. (1998) found that in plants, the N-end ruleincludes basic and aromatic residues. It is a possibility thatproteolytic cleavage by plant proteases near the start of α-helix 3 ofsubject B.t. insecticidal proteins may expose a destabilizing N-terminalamino acid. Such processing may target the cleaved proteins for rapiddecay and limit the accumulation of the B.t. insecticidal proteins tolevels insufficient for effective insect control. Accordingly, forN-terminal deletion variants that begin with one of the destabilizingamino acids, applicants prefer to add a codon that specifies a G(glycine) amino acid between the translational initiation methionine andthe destabilizing amino acid.

Example 2 gives specific examples of amino-terminal deletion variants ofDIG-3 in accordance with the invention. Additional useful fragments thatretain toxicity can be identified by trypsin or chymotrypsin digestionof the full length solubilized crystal protein. Further examples oftoxic DIG-3 protein fragments may be encoded by fragments of the DIG-3coding region. Insect active DIG-3 variants will mostly have a shortN-terminal truncation and a long C-terminal truncation. The N-terminalend of the smallest toxic fragment is conveniently determined byN-terminal amino acid sequence determination of trypsin- orchymotrypsin-treated soluble crystal protein by techniques routinelyavailable in the art.

Chimeric Toxins. Chimeric proteins utilizing the core toxin segment ofone Cry toxin fused to the protoxin segment of another Cry toxin havepreviously been reported. DIG-3 variants include toxins comprising anN-terminal core toxin segment of a DIG-3 toxin (which may be full lengthor have the N-terminal deletions described above) fused to aheterologous protoxin segment at some point past the end of the coretoxin segment. The transition to the heterologous protoxin segment canoccur at approximately the native core toxin/protoxin junction or, inthe alternative, a portion of the native protoxin (extending past thecore toxin segment) can be retained with the transition to theheterologous protoxin occurring downstream. As an example, a chimerictoxin of the subject invention has the full core toxin segment of DIG-3(amino acids 1-643) and a heterologous protoxin segment (amino acids 643to the C-terminus). In a preferred embodiment, the heterologous protoxinsegment is derived from a Cry1Ab delta-endotoxin, as illustrated in SEQID NO:5.

SEQ ID NO:4 discloses the 545 amino acid sequence of a Cry1Ab protoxinsegment useful in DIG-3 variants of the invention. Attention is drawn tothe last about 100 to 150 amino acids of this protoxin segment, which itis most critical to include in the chimeric toxin of the subjectinvention.

Protease sensitivity variants. Insect gut proteases typically functionin aiding the insect in obtaining needed amino acids from dietaryprotein. The best understood insect digestive proteases are serineproteases, which appear to be the most common type (Englemann andGeraerts (1980), particularly in Lepidopteran species. Coleopteraninsects have guts that are more neutral to acidic than are Lepidopteranguts. The majority of Coleopteran larvae and adults, for exampleColorado potato beetle, have slightly acidic midguts, and cysteineproteases provide the major proteolytic activity (Wolfson and Murdock,1990). More precisely, Thie and Houseman (1990) identified andcharacterized the cysteine proteases, cathepsin B-like and cathepsinH-like, and the aspartyl protease, cathepsin D-like, in Colorado potatobeetle. Gillikin et al. (1992) characterized the proteolytic activity inthe guts of western corn rootworm larvae and found primarily cysteineproteases. U.S. Pat. No. 7,230,167 disclosed that a protease activityattributed to cathepsin G exists in western corn rootworm. The diversityand different activity levels of the insect gut proteases may influencean insect's sensitivity to a particular B.t. toxin.

In another embodiment of the invention, protease cleavage sites may beengineered at desired locations to affect protein processing within themidgut of susceptible larvae of certain insect pests. These proteasecleavage sites may be introduced by methods such as chemical genesynthesis or splice overlap PCR (Horton et al., 1989). Serine proteaserecognition sequences, for example, can optionally be inserted atspecific sites in the Cry protein structure to effect protein processingat desired deletion points within the midgut of susceptible larvae.Serine proteases that can be exploited in such fashion includeLepidopteran midgut serine proteases such as trypsin or trypsin-likeenzymes, chymotrypsin, elastase, etc. (Christeller et al., 1992).Further, deletion sites identified empirically by sequencing Cry proteindigestion products generated with unfractionated larval midgut proteasepreparations or by binding to brush border membrane vesicles can beengineered to effect protein activation. Modified Cry proteins generatedeither by gene deletion or by introduction of protease cleavage siteshave improved activity on Lepidopteran pests including Ostrinianubilalis, Diatraea grandiosella, Helicoverpa zea, Agrotis ipsilon,Spodoptera frugiperda, Spodoptera exigua, Diatraea saccharalis,Loxagrotis albicosta, and other target pests.

Coleopteran serine proteases such as trypsin, chymotrypsin and cathepsinG-like protease, Coleopteran cysteine proteases such as cathepsins(B-like, L-like, O-like, and K-like proteases) (Koiwa et al., (2000) andBown et al., (2004)], Coleopteran metalloproteases such as ADAM10[Ochoa-Campuzano et al., (2007)), and Coleopteran aspartic acidproteases such as cathepsins D-like and E-like, pepsin, plasmepsin, andchymosin may further be exploited by engineering appropriate recognitionsequences at desired processing sites to affect Cry protein processingwithin the midgut of susceptible larvae of certain insect pests.

A preferred location for the introduction of such protease cleavagesites may be within the “spacer” region between α-helix 2B and α-helix3, for example within amino acids 109 to 113 of the full length DIG-3protein (SEQ ID NO:2 and Table 1). Modified Cry proteins generatedeither by gene deletion or by introduction of protease cleavage siteshave improved activity on insect pests including but not limited towestern corn rootworm, southern corn root worn, northern corn rootworm,and the like.

Various technologies exist to enable determination of the sequence ofthe amino acids which comprise the N-terminal or C-terminal residues ofpolypeptides. For example, automated Edman degradation methodology canbe used in sequential fashion to determine the N-terminal amino acidsequence of up to 30 amino acid residues with 98% accuracy per residue.Further, determination of the sequence of the amino acids comprising thecarboxy end of polypeptides is also possible [Bailey et al., (1992);U.S. Pat. No. 6,046,053]. Thus, in some embodiments, B.t. Cry proteinswhich have been activated by means of proteolytic processing, forexample, by proteases prepared from the gut of an insect, may becharacterized and the N-terminal or C-terminal amino acids of theactivated toxin fragment identified. DIG-3 variants produced byintroduction or elimination of protease processing sites at appropriatepositions in the coding sequence to allow, or eliminate, proteolyticcleavage of a larger variant protein by insect, plant or microorganismproteases are within the scope of the invention. The end result of suchmanipulation is understood to be the generation of toxin fragmentmolecules having the same or better activity as the intact (full length)toxin protein.

Domains of the DIG-3 toxin. The separate domains of the DIG-3 toxin,(and variants that are 90%, 95%, or 97% identical to such domains) areexpected to be useful in forming combinations with domains from otherCry toxins to provide new toxins with increased spectrum of pesttoxicity, improved potency, or increased protein stability. Domain I ofthe DIG-3 protein consists of amino acid residues 56 to 278 of SEQ IDNO:2. Domain II of the DIG-3 protein consists of amino acid residues 283to 493 of SEQ ID NO:2. Domain III of the DIG-3 protein consists of aminoacid residues 503 to 641 of SEQ ID NO:2. Domain swapping or shuffling isa mechanism for generating altered delta-endotoxin proteins. Domains IIand III may be swapped between delta-endotoxin proteins, resulting inhybrid or chimeric toxins with improved pesticidal activity or targetspectrum. Domain II is involved in receptor binding, and the DIG-3Domain II is very divergent from other Cry1B toxins. Domain III bindscertain classes of receptor proteins and perhaps participates ininsertion of an oligomeric toxin pre-pore. Some Domain III substitutionsin other toxins have been shown to produce superior toxicity againstSpodoptera exigua (de Maagd et al., 1996), and guidance exists on thedesign of the Cry toxin domain swaps (Knight et al., 2004).

Methods for generating recombinant proteins and testing them forpesticidal activity are well known in the art (see, for example, Naimovet al., (2001), de Maagd et al., (1996), Ge et al., (1991), Schnepf etal., (1990), Rang et al., (1999)). Domain I from Cry1A and Cry3Aproteins has been studied for the ability to insert and form pores inmembranes. α-helix 4 and α-helix 5 of Domain I play key roles inmembrane insertion and pore formation [Walters et al., (1993), Gazit etal., (1998); Nunez-Valdez et al., (2001)], with the other alpha helicesproposed to contact the membrane surface like the ribs of an umbrella(Bravo et al., (2007); Gazit et al., (1998)).

DIG-3 variants created by making a limited number of amino aciddeletions, substitutions, or additions. Amino acid deletions,substitutions, and additions to the amino acid sequence of SEQ ID NO:2can readily be made in a sequential manner and the effects of suchvariations on insecticidal activity can be tested by bioassay. Providedthe number of changes is limited in number, such testing does notinvolve unreasonable experimentation. The invention includesinsecticidally active variants of the core toxin segment (amino acids1-643 of SEQ ID NO:2, or amino acids 73-643 of SEQ ID NO:2) in which upto 10, up to 15, or up to 20 independent amino acid additions,deletions, or substitutions have been made.

The invention includes DIG-3 variants having a core toxin segment thatis 90%, 95% or 97% identical to amino acids 1-643 of SEQ ID NO:2 oramino acids 73-643 of SEQ ID NO:2.

Variants may be made by making random mutations or the variants may bedesigned. In the case of designed mutants, there is a high probabilityof generating variants with similar activity to the native toxin whenamino acid identity is maintained in critical regions of the toxin whichaccount for biological activity or are involved in the determination ofthree-dimensional configuration which ultimately is responsible for thebiological activity. A high probability of retaining activity will alsooccur if substitutions are conservative. Amino acids may be placed inthe following classes: non-polar, uncharged polar, basic, and acidic.Conservative substitutions whereby an amino acid of one class isreplaced with another amino acid of the same type are least likely tomaterially alter the biological activity of the variant. Table 2provides a listing of examples of amino acids belonging to each class.

TABLE 2 Class of Amino Acid Examples of Amino Acids Nonpolar Side ChainsAla (A), Val (V), Leu (L), Ile (I), Pro (P), Met (M), Phe (F), Trp (W)Uncharged Polar Side Chains Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y),Asn (N), Gln (Q) Acidic Side Chains Asp (D), Glu (E) Basic Side ChainsLys (K), Arg (R), His (H) Beta-branched Side Chains Thr, Val, IleAromatic Side Chains Tyr, Phe, Trp, His

In some instances, non-conservative substitutions can also be made. Thecritical factor is that these substitutions must not significantlydetract from the biological activity of the toxin. Variants includepolypeptides that differ in amino acid sequence due to mutagenesis.Variant proteins encompassed by the present invention are biologicallyactive, that is they continue to possess the desired biological activityof the native protein, namely, retaining pesticidal activity.

Variant proteins can also be designed that differ at the sequence levelbut that retain the same or similar overall essential three-dimensionalstructure, surface charge distribution, and the like. See e.g. U.S. Pat.No. 7,058,515; Larson et al. (2002); Stemmer (1994a,1994b, 1995); andCrameri et al. (1996a, 1996b, 1997).

Nucleic Acids. Isolated nucleic acids encoding DIG-3 toxins are oneaspect of the present invention. This includes nucleic acids encodingSEQ ID NO:2 and SEQ ID NO:5, and complements thereof, as well as othernucleic acids that encode insecticidal variants of SEQ ID NO:2. Becauseof the redundancy of the genetic code, a variety of different DNAsequences can encode the amino acid sequences disclosed herein. It iswell within the skill of a person trained in the art to create thesealternative DNA sequences encoding the same, or essentially the same,toxins.

Gene synthesis. DNA sequences encoding the improved Cry proteinsdescribed herein can be made by a variety of methods well-known in theart. For example, synthetic gene segments and synthetic genes can bemade by phosphite tri-ester and phosphoramidite chemistry (Caruthers etal., 1987), and commercial vendors are available to perform DNAsynthesis on demand. Sequences encoding full-length DIG-3 proteins canbe assembled in a variety of ways including, for example, by ligation ofrestriction fragments or polymerase chain reaction assembly ofoverlapping oligonucleotides (Stewart and Burgin, 2005). Further,sequences encoding terminal deletions can be made by PCR amplificationusing site-specific terminal oligonucleotides.

Nucleic acids encoding DIG-3 toxins can be made for example, bysynthetic construction by methods currently practiced by any of severalcommercial suppliers. (See for example, U.S. Pat. No. 7,482,119 B2).These nucleic acids, or portions or variants thereof, may also beconstructed synthetically, for example, by use of a gene synthesizer andthe design methods of, for example, U.S. Pat. No. 5,380,831.Alternatively, variations of synthetic or naturally occurring genes maybe readily constructed using standard molecular biological techniquesfor making point mutations. Fragments of these genes can also be madeusing commercially available exonucleases or endonucleases according tostandard procedures. For example, enzymes such as Bal31 or site-directedmutagenesis can be used to systematically cut off nucleotides from theends of these genes. Also, gene fragments which encode active toxinfragments may be obtained using a variety of restriction enzymes.

Given the amino acid sequence for a DIG-3 toxin, a coding sequence canbe designed by reverse translating the coding sequence using codonspreferred by the intended host, and then refining the sequence usingalternative codons to remove sequences that might cause problems andprovide periodic stop codons to eliminate long open coding sequences inthe non-coding reading frames.

Quantifying Sequence Identity. To determine the percent identity of twoamino acid sequences or of two nucleic acid sequences, the sequences arealigned for optimal comparison purposes. The percent identity betweenthe two sequences is a function of the number of identical positionsshared by the sequences (i.e. percent identity=number of identicalpositions/total number of positions (e.g. overlapping positions)×100).In one embodiment, the two sequences are the same length. The percentidentity between two sequences can be determined using techniquessimilar to those described below, with or without allowing gaps. Incalculating percent identity, typically exact matches are counted.

The determination of percent identity between two sequences can beaccomplished using a mathematical algorithm. A nonlimiting example ofsuch an algorithm is that of Karlin and Altschul (1990), modified as inKarlin and Altschul (1993), and incorporated into the BLASTN and BLASTXprograms. BLAST searches may be conveniently used to identify sequenceshomologous (similar) to a query sequence in nucleic or proteindatabases. BLASTN searches can be performed, (score=100, word length=12)to identify nucleotide sequences having homology to claimed nucleic acidmolecules of the invention. BLASTX searches can be performed (score=50,word length=3) to identify amino acid sequences having homology toclaimed insecticidal protein molecules of the invention.

Gapped BLAST (Altschul et al., 1997) can be utilized to obtain gappedalignments for comparison purposes, Alternatively, PSI-Blast can be usedto perform an iterated search that detects distant relationships betweenmolecules (Altschul et al., 1997). When utilizing BLAST, Gapped BLAST,and PSI-Blast programs, the default parameters of the respectiveprograms can be used. See www.ncbi.nlm.nih.gov.

A non-limiting example of a mathematical algorithm utilized for thecomparison of sequences is the ClustalW algorithm (Thompson et al.,1994). ClustalW compares sequences and aligns the entirety of the aminoacid or DNA sequence, and thus can provide data about the sequenceconservation of the entire amino acid sequence or nucleotide sequence.The ClustalW algorithm is used in several commercially availableDNA/amino acid analysis software packages, such as the ALIGNX module ofthe Vector NTI Program Suite (Invitrogen, Inc., Carlsbad, Calif.). Whenaligning amino acid sequences with ALIGNX, one may conveniently use thedefault settings with a Gap open penalty of 10, a Gap extend penalty of0.1 and the blosum63mt2 comparison matrix to assess the percent aminoacid similarity (consensus) or identity between the two sequences. Whenaligning DNA sequences with ALIGNX, one may conveniently use the defaultsettings with a Gap open penalty of 15, a Gap extend penalty of 6.6 andthe swgapdnamt comparison matrix to assess the percent identity betweenthe two sequences.

Another non-limiting example of a mathematical algorithm utilized forthe comparison of sequences is that of Myers and Miller (1988). Such analgorithm is incorporated into the wSTRETCHER program, which is part ofthe wEMBOSS sequence alignment software package (available athttp://emboss.sourceforge.net/). wSTRETCHER calculates an optimal globalalignment of two sequences using a modification of the classic dynamicprogramming algorithm which uses linear space. The substitution matrix,gap insertion penalty and gap extension penalties used to calculate thealignment may be specified. When utilizing the wSTRETCHER program forcomparing nucleotide sequences, a Gap open penalty of 16 and a Gapextend penalty of 4 can be used with the scoring matrix file EDNAFULL.When used for comparing amino acid sequences, a Gap open penalty of 12and a Gap extend penalty of 2 can be used with the EBLOSUM62 scoringmatrix file.

A further non-limiting example of a mathematical algorithm utilized forthe comparison of sequences is that of Needleman and Wunsch (1970),which is incorporated in the sequence alignment software packages GAPVersion 10 and wNEEDLE (http://emboss.sourceforge.net/). GAP Version 10may be used to determine sequence identity or similarity using thefollowing parameters: for a nucleotide sequence, % identity and %similarity are found using GAP Weight of 50 and Length Weight of 3, andthe nwsgapdna. cmp scoring matrix. For amino acid sequence comparison, %identity or % similarity are determined using GAP weight of 8 and lengthweight of 2, and the BLOSUM62 scoring program.

wNEEDLE reads two input sequences, finds the optimum alignment(including gaps) along their entire length, and writes their optimalglobal sequence alignment to file. The algorithm explores all possiblealignments and chooses the best, using .a scoring matrix that containsvalues for every possible residue or nucleotide match. wNEEDLE finds thealignment with the maximum possible score, where the score of analignment is equal to the sum of the matches taken from the scoringmatrix, minus penalties arising from opening and extending gaps in thealigned sequences. The substitution matrix and gap opening and extensionpenalties are user-specified. When amino acid sequences are compared, adefault Gap open penalty of 10, a Gap extend penalty of 0.5, and theEBLOSUM62 comparison matrix are used. When DNA sequences are comparedusing wNEEDLE, a Gap open penalty of 10, a Gap extend penalty of 0.5,and the EDNAFULL comparison matrix are used.

Equivalent programs may also be used. By “equivalent program” isintended any sequence comparison program that, for any two sequences inquestion, generates an alignment having identical nucleotide or aminoacid residue matches and an identical percent sequence identity whencompared to the corresponding alignment generated by ALIGNX, wNEEDLE, orwSTRETCHER. The % identity is the percentage of identical matchesbetween the two sequences over the reported aligned region (includingany gaps in the length) and the % similarity is the percentage ofmatches between the two sequences over the reported aligned region(including any gaps in the length).

Alignment may also be performed manually by inspection.

Recombinant hosts. The toxin-encoding genes of the subject invention canbe introduced into a wide variety of microbial or plant hosts.Expression of the toxin gene results, directly or indirectly, in theintracellular production and maintenance of the pesticidal protein. Withsuitable microbial hosts, e.g. Pseudomonas, the microbes can be appliedto the environment of the pest, where they will proliferate and beingested. The result is a control of the pest. Alternatively, themicrobe hosting the toxin gene can be treated under conditions thatprolong the activity of the toxin and stabilize the cell. The treatedcell, which retains the toxic activity, then can be applied to theenvironment of the target pest.

Where the B.t. toxin gene is introduced via a suitable vector into amicrobial host, and said host is applied to the environment in a livingstate, it is essential that certain host microbes be used. Microorganismhosts are selected which are known to occupy the “phytosphere”(phylloplane, phyllosphere, rhizosphere, and/or rhizoplane) of one ormore crops of interest. These microorganisms are selected so as to becapable of successfully competing in the particular environment (cropand other insect habitats) with the wild-type indigenous microorganisms,provide for stable maintenance and expression of the gene expressing thepolypeptide pesticide, and, desirably, provide for improved protectionof the pesticide from environmental degradation and inactivation.

A large number of microorganisms are known to inhabit the phylloplane(the surface of the plant leaves) and/or the rhizosphere (the soilsurrounding plant roots) of a wide variety of important crops. Thesemicroorganisms include bacteria, algae, and fungi. Of particularinterest are microorganisms, such as bacteria, e.g. genera Pseudomonas,Erwinia, Serratia, Klebsiella, Xanthomonas, Streptomyces, Rhizobium,Sinorhizobium, Rhodopseudomonas, Methylophilius, Agrobacterium,Acetobacter, Lactobacillus, Arthrobacter, Azotobacter, Leuconostoc, andAlcaligenes; fungi, particularly yeast, e.g. genera Saccharomyces,Cryptococcus, Kluyveromyces, Sporobolomyces, Rhodotorula, andAureobasidium. Of particular interest are such phytosphere bacterialspecies as Pseudomonas syringae, Pseudomonas fluorescens, Serratiamarcescens, Acetobacter xylinum, Agrobacterium tumefaciens,Agrobacterium radiobacter, Rhodopseudomonas spheroides, Xanthomonascampestris, Sinorhizobium meliloti (formerly Rhizobium meliloti),Alcaligenes eutrophus, and Azotobacter vinelandii; and phytosphere yeastspecies such as Rhodotorula rubra, R. glutinis, R. marina, R.aurantiaca, Cryptococcus albidus, C. diffluens, C. laurentii,Saccharomyces rosei, S. pretoriensis, S. cerevisiae, Sporobolomycesroseus, S. odorus, Kluyveromyces veronae, and Aureobasidium pollulans.Of particular interest are the pigmented microorganisms.

Methods of Controlling Insect Pests

When an insect comes into contact with an effective amount of toxindelivered via transgenic plant expression, formulated proteincompositions(s), sprayable protein composition(s), a bait matrix orother delivery system, the results are typically death of the insect, orthe insects do not feed upon the source which makes the toxins availableto the insects.

The subject protein toxins can be “applied” or provided to contact thetarget insects in a variety of ways. For example, transgenic plants(wherein the protein is produced by and present in the plant) can beused and are well-known in the art. Expression of the toxin genes canalso be achieved selectively in specific tissues of the plants, such asthe roots, leaves, etc. This can be accomplished via the use oftissue-specific promoters, for example. Spray-on applications areanother example and are also known in the art. The subject proteins canbe appropriately formulated for the desired end use, and then sprayed(or otherwise applied) onto the plant and/or around the plant/to thevicinity of the plant to be protected—before an infestation isdiscovered, after target insects are discovered, both before and after,and the like. Bait granules, for example, can also be used and are knownin the art.

Transgenic Plants

The subject proteins can be used to protect practically any type ofplant from damage by a Lepidopteran insect. Nonlimiting examples of suchplants include maize, sunflower, soybean, cotton, canola, rice, sorghum,wheat, barley, vegetables, ornamentals, peppers (including hot peppers),sugar beets, fruit, and turf, to name but a few. Methods fortransforming plants are well known in the art, and illustrativetransformation methods are described in the Examples.

A preferred embodiment of the subject invention is the transformation ofplants with genes encoding the subject insecticidal protein or itsvariants. The transformed plants are resistant to attack by an insecttarget pest by virtue of the presence of controlling amounts of thesubject insecticidal protein or its variants in the cells of thetransformed plant. By incorporating genetic material that encodes theinsecticidal properties of the B.t. insecticidal toxins into the genomeof a plant eaten by a particular insect pest, the adult or larvae woulddie after consuming the food plant. Numerous members of themonocotyledonous and dicotyledonous classifications have beentransformed. Transgenic agronomic crops as well as fruits and vegetablesare of commercial interest. Such crops include, but are not limited to,maize, rice, soybeans, canola, sunflower, alfalfa, sorghum, wheat,cotton, peanuts, tomatoes, potatoes, and the like. Several techniquesexist for introducing foreign genetic material into plant cells, and forobtaining plants that stably maintain and express the introduced gene.Such techniques include acceleration of genetic material coated ontomicroparticles directly into cells (U.S. Pat. No. 4,945,050 and U.S.Pat. No. 5,141,131). Plants may be transformed using Agrobacteriumtechnology, see U.S. Pat. No. 5,177,010, U.S. Pat. No. 5,104,310,European Patent Application No. 0131624B1, European Patent ApplicationNo. 120516, European Patent Application No. 159418B1, European PatentApplication No. 176112, U.S. Pat. No. 5,149,645, U.S. Pat. No.5,469,976, U.S. Pat. No. 5,464,763, U.S. Pat. No. 4,940,838, U.S. Pat.No. 4,693,976, European Patent Application No. 116718, European PatentApplication No. 290799, European Patent Application No. 320500, EuropeanPatent Application No. 604662, European Patent Application No. 627752,European Patent Application No. 0267159, European Patent Application No.0292435, U.S. Pat. No. 5,231,019, U.S. Pat. No. 5,463,174, U.S. Pat. No.4,762,785, U.S. Pat. No. 5,004,863, and U.S. Pat. No. 5,159,135. Othertransformation technology includes WHISKERS™ technology, see U.S. Pat.No. 5,302,523 and U.S. Pat. No. 5,464,765. Electroporation technologyhas also been used to transform plants, see WO 87/06614, U.S. Pat. No.5,472,869, U.S. Pat. No. 5,384,253, WO 9209696, and WO 9321335. All ofthese transformation patents and publications are incorporated byreference. In addition to numerous technologies for transforming plants,the type of tissue which is contacted with the foreign genes may vary aswell. Such tissue would include but would not be limited to embryogenictissue, callus tissue types I and II, hypocotyl, meristem, and the like.Almost all plant tissues may be transformed during dedifferentiationusing appropriate techniques within the skill of an artisan.

Genes encoding DIG-3 toxins can be inserted into plant cells using avariety of techniques which are well known in the art as disclosedabove. For example, a large number of cloning vectors comprising amarker that permits selection of the transformed microbial cells and areplication system functional in Escherichia coli are available forpreparation and modification of foreign genes for insertion into higherplants. Such manipulations may include, for example, the insertion ofmutations, truncations, additions, or substitutions as desired for theintended use. The vectors comprise, for example, pBR322, pUC series, M13mp series, pACYC184, etc. Accordingly, the sequence encoding the Cryprotein or variants can be inserted into the vector at a suitablerestriction site. The resulting plasmid is used for transformation ofcells of E. coli, the cells of which are cultivated in a suitablenutrient medium, then harvested and lysed so that workable quantities ofthe plasmid are recovered. Sequence analysis, restriction fragmentanalysis, electrophoresis, and other biochemical-molecular biologicalmethods are generally carried out as methods of analysis. After eachmanipulation, the DNA sequence used can be cleaved and joined to thenext DNA sequence. Each manipulated DNA sequence can be cloned in thesame or other plasmids.

The use of T-DNA-containing vectors for the transformation of plantcells has been intensively researched and sufficiently described in EP120516; Lee and Gelvin (2008), Fraley et al. (1986), and An et al.(1985), and is well established in the field.

Once the inserted DNA has been integrated into the plant genome, it isrelatively stable throughout subsequent generations. The vector used totransform the plant cell normally contains a selectable marker geneencoding a protein that confers on the transformed plant cellsresistance to a herbicide or an antibiotic, such as bialaphos,kanamycin, G418, bleomycin, or hygromycin, inter alfa. The individuallyemployed selectable marker gene should accordingly permit the selectionof transformed cells while the growth of cells that do not contain theinserted DNA is suppressed by the selective compound.

A large number of techniques are available for inserting DNA into a hostplant cell. Those techniques include transformation with T-DNA deliveredby Agrobacterium tumefaciens or Agrobacterium rhizogenes as thetransformation agent. Additionally, fusion of plant protoplasts withliposomes containing the DNA to be delivered, direct injection of theDNA, biolistics transformation (microparticle bombardment), orelectroporation, as well as other possible methods, may be employed.

In a preferred embodiment of the subject invention, plants will betransformed with genes wherein the codon usage of the protein codingregion has been optimized for plants. See, for example, U.S. Pat. No.5,380,831, which is hereby incorporated by reference. Also,advantageously, plants encoding a truncated toxin will be used. Thetruncated toxin typically will encode about 55% to about 80% of the fulllength toxin. Methods for creating synthetic B.t. genes for use inplants are known in the art (Stewart, 2007).

Regardless of transformation technique, the gene is preferablyincorporated into a gene transfer vector adapted to express the B.tinsecticidal toxin genes and variants in the plant cell by including inthe vector a plant promoter. In addition to plant promoters, promotersfrom a variety of sources can be used efficiently in plant cells toexpress foreign genes. For example, one may use promoters of bacterialorigin, such as the octopine synthase promoter, the nopaline synthasepromoter, and the mannopine synthase promoter. Promoters of plant virusorigin may be used, for example, the 35S and 19S promoters ofCauliflower Mosaic Virus, a promoter from Cassaya Vein Mosaic Virus, andthe like. Plant promoters include, but are not limited to,ribulose-1,6-bisphosphate (RUBP) carboxylase small subunit (ssu),beta-conglycinin promoter, phaseolin promoter, ADH (alcoholdehydrogenase) promoter, heat-shock promoters, ADF (actindepolymerization factor) promoter, ubiquitin promoter, actin promoter,and tissue specific promoters. Promoters may also contain certainenhancer sequence elements that may improve the transcriptionefficiency. Typical enhancers include but are not limited to ADH1-intron1 and ADH1-intron 6. Constitutive promoters may be used. Constitutivepromoters direct continuous gene expression in nearly all cells typesand at nearly all times (e.g., actin, ubiquitin, CaMV 35S). Tissuespecific promoters are responsible for gene expression in specific cellor tissue types, such as the leaves or seeds (e.g. zein, oleosin, napin,ACP (Acyl Carrier Protein) promoters), and these promoters may also beused. Promoters may also be used that are active during a certain stageof the plants' development as well as active in specific plant tissuesand organs. Examples of such promoters include but are not limited topromoters that are root specific, pollen-specific, embryo specific, cornsilk specific, cotton fiber specific, seed endosperm specific, phloemspecific, and the like.

Under certain circumstances it may be desirable to use an induciblepromoter. An inducible promoter is responsible for expression of genesin response to a specific signal, such as: physical stimulus (e.g. heatshock genes); light (e.g. RUBP carboxylase); hormone (e.g.glucocorticoid); antibiotic (e.g. tetracycline); metabolites; and stress(e.g. drought). Other desirable transcription and translation elementsthat function in plants may be used, such as 5′ untranslated leadersequences, RNA transcription termination sequences and poly-adenylateaddition signal sequences. Numerous plant-specific gene transfer vectorsare known to the art.

Transgenic crops containing insect resistance (IR) traits are prevalentin corn and cotton plants throughout North America, and usage of thesetraits is expanding globally. Commercial transgenic crops combining IRand herbicide tolerance (HT) traits have been developed by multiple seedcompanies. These include combinations of IR traits conferred by B.t.insecticidal proteins and HT traits such as tolerance to AcetolactateSynthase (ALS) inhibitors such as sulfonylureas, imidazolinones,triazolopyrimidine, sulfonanilides, and the like, Glutamine Synthetase(GS) inhibitors such as bialaphos, glufosinate, and the like,4-HydroxyPhenylPyruvate Dioxygenase (HPPD) inhibitors such asmesotrione, isoxaflutole, and the like,5-EnolPyruvylShikimate-3-Phosphate Synthase (EPSPS) inhibitors such asglyphosate and the like, and Acetyl-Coenzyme A Carboxylase (ACCase)inhibitors such as haloxyfop, quizalofop, diclofop, and the like. Otherexamples are known in which transgenically provided proteins provideplant tolerance to herbicide chemical classes such as phenoxy acidsherbicides and pyridyloxyacetates auxin herbicides (see WO 2007/053482A2), or phenoxy acids herbicides and aryloxyphenoxypropionatesherbicides (see WO 2005107437 A2, A3). The ability to control multiplepest problems through IR traits is a valuable commercial productconcept, and the convenience of this product concept is enhanced ifinsect control traits and weed control traits are combined in the sameplant. Further, improved value may be obtained via single plantcombinations of IR traits conferred by a B.t. insecticidal protein suchas that of the subject invention, with one or more additional HT traitssuch as those mentioned above, plus one or more additional input traits(e.g. other insect resistance conferred by B.t.-derived or otherinsecticidal proteins, insect resistance conferred by mechanisms such asRNAi and the like, nematode resistance, disease resistance, stresstolerance, improved nitrogen utilization, and the like), or outputtraits (e.g. high oils content, healthy oil composition, nutritionalimprovement, and the like). Such combinations may be obtained eitherthrough conventional breeding (breeding stack) or jointly as a noveltransformation event involving the simultaneous introduction of multiplegenes (molecular stack). Benefits include the ability to manage insectpests and improved weed control in a crop plant that provides secondarybenefits to the producer and/or the consumer. Thus, the subjectinvention can be used in combination with other traits to provide acomplete agronomic package of improved crop quality with the ability toflexibly and cost effectively control any number of agronomic issues.

Target Pests

The DIG-3 toxins of the invention are particularly suitable for use incontrol of Lepidopteran insects. Lepidopterans are an important group ofagricultural, horticultural, and household pests which cause a verylarge amount of damage each year. This insect order encompasses foliar-and root-feeding larvae and adults. Lepidopteran insect pests include,but are not limited to: Achoroia grisella, Acleris gloverana, Aclerisvariana, Adoxophyes orana, Agrotis Ipsilon (black cutworm), Alabamaargillacea, Alsophila pometaria, Amyelois transitella, Anagastakuehniella, Anarsia lineatella, Anisota senatoria, Antheraea pernyi,Anticarsia gemmatalis, Archips sp., Argyrotaenia sp., Athetis mindara,Bombyx mori, Bucculatrix thurberiella, Cadra cautella, Choristoneurasp., Cochylls hospes, Colias eurytheme, Corcyra cephalonica, Cydialatiferreanus, Cydia pomonella, Datana integerrima, Dendrolimussibericus, Desmia feneralis, Diaphania hyalinata, Diaphania nitidalis,Diatraea grandiosella (southwestern corn borer), Diatraea saccharalis(sugarcane borer), Ennomos subsignaria, Eoreuma loftini, Esphestiaelutella, Erannis tilaria, Estigmene acrea, Eulia salubricola,Eupocoellia ambiguella, Eupoecilia ambiguella, Euproctis chrysorrhoea,Euxoa messoria, Galleria mellonella, Grapholita molesta, Harrisinaamericana, Helicoverpa subflexa, Helicoverpa zea (corn earworm),Heliothis virescens (tobacco budworm), Hemileuca oliviae, Homoeosomaelectellum (sunflower head moth), Hyphantia cunea, Keiferialycopersicella, Lambdina fiscellaria fiscellaria, Lambdina fiscellarialugubrosa, Leucoma salicis, Lobesia botrana, Loxagrotis albicosta(western bean cutworm), Loxostege sticticalis, Lymantria dispar, Macallathyrisalis, Malacosoma sp., Mamestra brassicae, Mamestra configurata(bertha armyworm), Manduca quinquemaculata, Manduca sexta (tobaccohornworm), Maruca testulalis, Melanchra picta, Operophtera brumata,Orgyia sp., Ostrinia nubilalis (European corn borer), Paleacritavernata, Papiapema nebris (common stalk borer), Papilio cresphontes,Pectinophora gossypiella, Phryganidia californica, Phyllonorycterblancardella, Pieris napi, Pieris rapae, Plathypena scabra, Platynotaflouendana, Platynota stultana, Platyptilia carduidactyla, Plodiainterpunctella, Plutella xylostella (diamondback moth), Pontiaprotodice, Pseudaletia unipuncta (armyworm), Pseudoplasia includens,Rachiplusia nu (Argentine looper), Sabulodes aegrotata, Schizuraconcinna, Sitotroga cerealella, Spilonta ocellana, Spodoptera frugiperda(fall armyworm), Spodoptera exigua (beet armyworm), Thaurnstopoeapityocampa, Ensola bisselliella, Trichoplusia hi, Udea rubigalis,Xylomyges curiails, and Yponomeuta padella.

Use of DIG-3 toxins to control Coleopteran pests of crop plants is alsocontemplated. In some embodiments, Cry proteins may be economicallydeployed for control of insect pests that include but are not limitedto, for example, rootworms such as Diabrotica undecimpunctata howardi(southern corn rootworm), Diabrotica longicornis barberi (northern cornrootworm), and Diabrotica virgifera (western corn rootworm), and grubssuch as the larvae of Cyclocephala borealis (northern masked chafer),Cyclocephala immaculate (southern masked chafer), and Popillia japonica(Japanese beetle).

Use of the DIG-3 toxins to control parasitic nematodes including, butnot limited to, root knot nematode (Meloidogyne icognita) and soybeancyst nematode (Heterodera glycines) is also contemplated.

Antibody Detection of DIG-3 Toxins

Anti-toxin antibodies. Antibodies to the toxins disclosed herein, or toequivalent toxins, or fragments of these toxins, can readily be preparedusing standard procedures in this art. Such antibodies are useful todetect the presence of the DIG-3 toxins.

Once the B.t. insecticidal toxin has been isolated, antibodies specificfor the toxin may be raised by conventional methods that are well knownin the art. Repeated injections into a host of choice over a period ofweeks or months will elicit an immune response and result in significantanti-B.t toxin serum titers. Preferred hosts are mammalian species andmore highly preferred species are rabbits, goats, sheep and mice. Blooddrawn from such immunized animals may be processed by establishedmethods to obtain antiserum (polyclonal antibodies) reactive with theB.t. insecticidal toxin. The antiserum may then be affinity purified byadsorption to the toxin according to techniques known in the art.Affinity purified antiserum may be further purified by isolating theimmunoglobulin fraction within the antiserum using procedures known inthe art. The resulting material will be a heterogeneous population ofimmunoglobulins reactive with the B.t. insecticidal toxin.

Anti-B.t. toxin antibodies may also be generated by preparing asemi-synthetic immunogen consisting of a synthetic peptide fragment ofthe B.t. insecticidal toxin conjugated to an immunogenic carrier.Numerous schemes and instruments useful for making peptide fragments arewell known in the art. Many suitable immunogenic carriers such as bovineserum albumin or keyhole limpet hemocyanin are also well known in theart, as are techniques for coupling the immunogen and carrier proteins.Once the semi-synthetic immunogen has been constructed, the procedurefor making antibodies specific for the B.t. insecticidal toxin fragmentis identical to those used for making antibodies reactive with naturalB.t. toxin.

Anti-B.t. toxin monoclonal antibodies (MAbs) are readily prepared usingpurified B.t. insecticidal toxin. Methods for producing MAbs have beenpracticed for over 20 years and are well known to those of ordinaryskill in the art. Repeated intraperitoneal or subcutaneous injections ofpurified B.t. insecticidal toxin in adjuvant will elicit an immuneresponse in most animals. Hyperimmunized B-lymphocytes are removed fromthe animal and fused with a suitable fusion partner cell line capable ofbeing cultured indefinitely. Preferred animals whose B-lymphocytes maybe hyperimmunized and used in the production of MAbs are mammals. Morepreferred animals are rats and mice and most preferred is the BALB/cmouse strain.

Numerous mammalian cell lines are suitable fusion partners for theproduction of hybridomas. Many such lines are available from theAmerican Type Culture Collection (ATCC, Manassas, Va.) and commercialsuppliers. Preferred fusion partner cell lines are derived from mousemyelomas and the HL-1® Friendly myeloma-653 cell line (Ventrex,Portland, Me.) is most preferred. Once fused, the resulting hybridomasare cultured in a selective growth medium for one to two weeks. Two wellknown selection systems are available for eliminating unfused myelomacells, or fusions between myeloma cells, from the mixed hybridomaculture. The choice of selection system depends on the strain of mouseimmunized and myeloma fusion partner used. The AAT selection system,described by Taggart and Samloff, (1983), may be used; however, the HAT(hypoxanthine, aminopterin, thymidine) selection system, described byLittlefield, (1964), is preferred because of its compatibility with thepreferred mouse strain and fusion partner mentioned above. Spent growthmedium is screened for immunospecific MAb secretion. Enzyme linkedimmunosorbent assay (ELISA) procedures are best suited for this purpose;though, radioimmunoassays adapted for large volume screening are alsoacceptable. Multiple screens designed to consecutively pare down theconsiderable number of irrelevant or less desired cultures may beperformed. Cultures that secrete MAbs reactive with the B.t.insecticidal toxin may be screened for cross-reactivity with known B.t.insecticidal toxins. MAbs that preferentially bind to the preferred B.t.insecticidal toxin may be isotyped using commercially available assays.Preferred MAbs are of the IgG class, and more highly preferred MAbs areof the IgG₁ and IgG_(2a) subisotypes.

Hybridoma cultures that secrete the preferred MAbs may be sub-clonedseveral times to establish monoclonality and stability. Well knownmethods for sub-cloning eukaryotic, non-adherent cell cultures includelimiting dilution, soft agarose and fluorescence activated cell sortingtechniques. After each subcloning, the resultant cultures preferably arere-assayed for antibody secretion and isotype to ensure that a stablepreferred MAb-secreting culture has been established.

The anti-B.t. toxin antibodies are useful in various methods ofdetecting the claimed B.t. insecticidal toxin of the instant invention,and variants or fragments thereof. It is well known that antibodieslabeled with a reporting group can be used to identify the presence ofantigens in a variety of milieus. Antibodies labeled with radioisotopeshave been used for decades in radioimmunoassays to identify, with greatprecision and sensitivity, the presence of antigens in a variety ofbiological fluids. More recently, enzyme labeled antibodies have beenused as a substitute for radiolabeled antibodies in the ELISA assay.Further, antibodies immunoreactive to the B.t. insecticidal toxin of thepresent invention can be bound to an immobilizing substance such as apolystyrene well or particle and used in immunoassays to determinewhether the B.t. toxin is present in a test sample.

Detection Using Nucleic Acid Probes

A further method for identifying the toxins and genes of the subjectinvention is through the use of oligonucleotide probes. These probes aredetectable nucleotide sequences. These sequences may be rendereddetectable by virtue of an appropriate radioactive label or may be madeinherently fluorescent as described in, for example, U.S. Pat. No.6,268,132. As is well known in the art, if the probe molecule andnucleic acid sample hybridize by forming strong base-pairing bondsbetween the two molecules, it can be reasonably assumed that the probeand sample have substantial sequence homology. Preferably, hybridizationis conducted under stringent conditions by techniques well-known in theart, as described, for example, in Keller and Manak (1993). Detection ofthe probe provides a means for determining in a known manner whetherhybridization has occurred. Such a probe analysis provides a rapidmethod for identifying toxin-encoding genes of the subject invention.The nucleotide segments which are used as probes according to theinvention can be synthesized using a DNA synthesizer and standardprocedures. These nucleotide sequences can also be used as PCR primersto amplify genes of the subject invention.

Nucleic Acid Hybridization

As is well known to those skilled in molecular biology, similarity oftwo nucleic acids can be characterized by their tendency to hybridize.As used herein the terms “stringent conditions” or “stringenthybridization conditions” are intended to refer to conditions underwhich a probe will hybridize (anneal) to its target sequence to adetectably greater degree than to other sequences (e.g. at least 2-foldover background). Stringent conditions are sequence-dependent and willbe different in different circumstances. By controlling the stringencyof the hybridization and/or washing conditions, target sequences thatare 100% complementary to the probe can be identified (homologousprobing). Alternatively, stringency conditions can be adjusted to allowsome mismatching in sequences so that lower degrees of similarity aredetected (heterologous probing). Generally, a probe is less than about1000 nucleotides in length, preferably less than 500 nucleotides inlength.

Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to pH 8.3 and thetemperature is at least about 30° C. for short probes (e.g. 10 to 50nucleotides) and at least about 60° C. for long probes (e.g. greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. Exemplary lowstringency conditions include hybridization with a buffer solution of30% to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulfate) at 37°C. and a wash in 1× to 2× SSC (20× SSC=3.0 M NaCl/0.3 M trisodiumcitrate) at 50° C. to 55° C. Exemplary moderate stringency conditionsinclude hybridization in 40% to 45% formamide, 1.0 M NaCl, 1% SDS at 37°C. and a wash in 0.5× to 1× SSC at 55° C. to 60° C. Exemplary highstringency conditions include hybridization in 50% formamide, 1 M NaCl,1% SDS at 37° C. and a wash in 0.1× SSC at 60° C. to 65° C. Optionally,wash buffers may comprise about 0.1% to about 1% SDS. Duration ofhybridization is generally less than about 24 hours, usually about 4 toabout 12 hours.

Specificity is typically the function of post-hybridization washes, thecritical factors being the ionic strength and temperature of the finalwash solution. For DNA/DNA hybrids, the thermal melting point (T_(m)) isthe temperature (under defined ionic strength and pH at which 50% of acomplementary target sequence hybridizes to a perfectly matched probe.T_(m) is reduced by about 1° C. for each 1% of mismatching; thus, T_(m),hybridization conditions, and/or wash conditions can be adjusted tofacilitate annealing of sequences of the desired identity. For example,if sequences with >90% identity are sought, the T_(m) can be decreased10° C. Generally, stringent conditions are selected to be about 5° C.lower than the T_(m) for the specific sequence and its complement at adefined ionic strength and pH. However, highly stringent conditions canutilize a hybridization and/or wash at 1° C., 2° C., 3° C., or 4° C.lower than the T_(m); moderately stringent conditions can utilize ahybridization and/or wash at 6° C., 7° C., 8° C., 9° C., or 10° C. lowerthan the T_(m), and low stringency conditions can utilize ahybridization and/or wash at 11° C., 12° C., 13° C., 14° C., 15° C., or20° C. lower than the T_(m).

T_(m) (in ° C.) may be experimentally determined or may be approximatedby calculation. For DNA-DNA hybrids, the T_(m) can be approximated fromthe equation of Meinkoth and Wahl (1984):

T _(m)(° C.)=81.5° C.+16.6(log M)+0.41(% GC)−0.61(% formamide)−500/L;

where M is the molarity of monovalent cations, % GC is the percentage ofguanosine and cytosine nucleotides in the DNA, % formamide is thepercentage of formamide in the hybridization solution, and L is thelength of the hybrid in base pairs.

Alternatively, the T_(m) is described by the following formula (Beltz etal., 1983).

T _(m)(° C.)=81.5° C.+16.6(log [Na+])+0.41(% GC)−0.61(% formamide)−600/L

where [Na+] is the molarity of sodium ions, % GC is the percentage ofguanosine and cytosine nucleotides in the DNA, % formamide is thepercentage of formamide in the hybridization solution, and L is thelength of the hybrid in base pairs.

Using the equations, hybridization and wash compositions, and desiredT_(m), those of ordinary skill will understand that variations in thestringency of hybridization and/or wash solutions are inherentlydescribed. If the desired degree of mismatching results in a T_(m) ofless than 45° C. (aqueous solution) or 32° C. (formamide solution), itis preferred to increase the SSC concentration so that a highertemperature can be used. An extensive guide to the hybridization ofnucleic acids is found in Tijssen (1993) and Ausubel et al. (1995) Alsosee Sambrook et al. (1989).

Hybridization of immobilized DNA on Southern blots with radioactivelylabeled gene-specific probes may be performed by standard methodsSambrook et al., supra.). Radioactive isotopes used for labelingpolynucleotide probes may include 32P, 33P, 14C, or 3H. Incorporation ofradioactive isotopes into polynucleotide probe molecules may be done byany of several methods well known to those skilled in the field ofmolecular biology. (See, e.g. Sambrook et al., supra.) In general,hybridization and subsequent washes may be carried out under stringentconditions that allow for detection of target sequences with homology tothe claimed toxin encoding genes. For double-stranded DNA gene probes,hybridization may be carried out overnight at 20°-25° C. below the T_(m)of the DNA hybrid in 6× SSPE, 5×Denhardt's Solution, 0.1% SDS, 0.1 mg/mLdenatured DNA [20× SSPE is 3 M NaCl, 0.2 M NaHPO₄, and 0.02 M EDTA(ethylenediamine tetra-acetic acid sodium salt); 100×Denhardt's Solutionis 20 gm/L Polyvinylpyrollidone, 20 gm/L Ficoll type 400 and 20 gm/LBovine Serum Albumin (fraction V)].

-   -   Washes may typically be carried out as follows:    -   Twice at room temperature for 15 minutes in 1× SSPE, 0.1% SDS        (low stringency wash).    -   Once at T_(m)-20° C. for 15 minutes in 0.2× SSPE, 0.1% SDS        (moderate stringency wash).

For oligonucleotide probes, hybridization may be carried out overnightat 10°-20° C. below the T_(m) of the hybrid in 6× SSPE, 5×Denhardt'ssolution, 0.1% SDS, 0.1 mg/mL denatured DNA. T_(m) for oligonucleotideprobes may be determined by the following formula (Suggs et al., 1981).

T _(m)(° C.)=2(number of T/A base pairs)+4(number of G/C base pairs)

Washes may typically be carried out as follows:

-   -   Twice at room temperature for 15 minutes 1× SSPE, 0.1% SDS (low        stringency wash).    -   Once at the hybridization temperature for 15 minutes in 1× SSPE,        0.1% SDS (moderate stringency wash).

Probe molecules for hybridization and hybrid molecules formed betweenprobe and target molecules may be rendered detectable by means otherthan radioactive labeling. Such alternate methods are intended to bewithin the scope of this invention.

All patents, patent applications, provisional applications, andpublications referred to or cited herein are incorporated by referencein their entirety to the extent they are not inconsistent with theexplicit teachings of this specification. Unless specifically indicatedor implied, the terms “a”, “an”, and “the” signify “at least one” asused herein. By the use of the term “genetic material” herein, it ismeant to include all genes, nucleic acid, DNA and RNA.

For designations of nucleotide residues of polynucleotides, DNA, RNA,oligonucleotides, and primers, and for designations of amino acidresidues of proteins, standard IUPAC abbreviations are employedthroughout this document. Nucleic acid sequences are presented in thestandard 5′ to 3′ direction, and protein sequences are presented in thestandard amino (N) terminal to carboxy (C) terminal direction.

Following are examples that illustrate procedures for practicing theinvention. It should be understood that the examples and embodimentsdescribed herein are for illustrative purposes only and that variousmodifications or changes in light thereof will be suggested to personsskilled in the art and are to be included within the spirit and purviewof this application and the scope of the appended claims. These examplesshould not be construed as limiting. All percentages are by weight andall solvent mixture proportions are by volume unless otherwise noted.All temperatures are in degrees Celsius.

Example 1 Isolation of a Gene Encoding DIG-3 Toxin

Nucleic acid encoding the insecticidal Cry protein designated herein asDIG-3 was isolated from genomic DNA of B.t. strain PS46L by PCR using adegenerate forward primer that hybridized to bases 1286 to 1311 of SEQID NO:1, and a mismatched reverse primer that hybridized to thecomplement of bases 2480 to 2499 of SEQ ID NO:1. This pair of primerswas used to amplify a fragment of 1214 bp, corresponding to nucleotides1286 to 2499 of SEQ ID NO:1. This sequence was used as the anchor pointto begin genome walking using methods adapted from the GenomeWalker™Universal Kit (Clontech, Palo Alto, Calif.). The nucleic acid sequenceof a fragment spanning the DIG-3 coding region was determined. SEQ IDNO:1 is the 3771 by nucleotide sequence encoding the full length DIG-3protein. SEQ ID NO:2 is the amino acid sequence of the full length DIG-3protein deduced from SEQ ID NO:1. It is noted that in Bacillus species,protein coding regions such as that of SEQ ID NO:1 may initiate with theTTG codon, which translationally represents the amino acid methionine.

Example 2 Deletion of Domain I α-Helices from DIG-3

To improve the insecticidal properties of the DIG-3 toxin, serial,step-wise deletions are made, each of which removes part of theN-terminus of the DIG-3 protein. The deletions remove part or all ofα-helix 1 and part or all of α-helix 2 in Domain I, while maintainingthe structural integrity of α-helix 3 through α-helix 7.

Deletions were designed as follows. This example utilizes the fulllength chimeric DNA sequence encoding the full-length DIG-3 protein e.g.SEQ ID NO:1 and SEQ ID NO:2, respectively) to illustrate the designprinciples with 67 specific variants. It utilizes the chimeric sequenceof SEQ ID NO:5 (DNA encoding DIG-3 core toxin segment fused to Cry1Abprotoxin segment) to provide an additional 67 specific variants. Oneskilled in the art will realize that other DNA sequences encoding all oran N-terminal portion of the DIG-3 protein may be similarly manipulatedto achieve the desired result. To devise the first deleted variantcoding sequence, all of the bases that encode α-helix 1 up to the codonfor the proline residue near the beginning of α-helix 2A (i.e. P73 forthe full length DIG-3 protein of SEQ ID NO:2), are removed. Thus,elimination of bases 1 to 216 of SEQ ID NO:1 removes the coding sequencefor amino acids 1 to 72 of SEQ ID NO:2. Reintroduction of a translationinitiating ATG (methionine) codon at the beginning (i.e. in front of thecodon corresponding to amino acid 73 of the full length protein)provides for the deleted variant coding sequence comprising an openreading frame of 3555 bases which encodes a deleted variant DIG-3protein comprising 1185 amino acids (i.e. methionine plus amino acids 73to 1256 of the full-length DIG-3 protein). Serial, stepwise deletionsthat remove additional codons for a single amino acid corresponding toresidues 73 to 112 of the full-length DIG-3 protein of SEQ ID NO:2provide variants lacking part or all of α-helix 2A and α-helix 2B. Thusa second designed deleted variant coding sequence requires eliminationof bases 1 to 219 of SEQ ID NO:1, thereby removing the coding sequencefor amino acids 1-73. Restoration of a functional open reading frame isagain accomplished by reintroduction of a translation initiationmethionine codon at the beginning of the remaining coding sequence, thusproviding for a second deleted variant coding sequence having an openreading frame of 3552 bases encoding a deleted variant DIG-3 proteincomprising 1184 amino acids (i.e. methionine plus amino acids 74 to 1256of the full-length DIG-3 protein). The last designed deleted variantcoding sequence requires removal of bases 1 to 336 of SEQ ID NO:1, thuseliminating the coding sequence for amino acids 1 to 112, and, afterreintroduction of a translation initiation methionine codon, providing adeletion variant coding sequence having an open reading frame of 3435bases which encodes a deletion variant DIG-3 protein of 1145 amino acids(i.e. methionine plus amino acids 113 to 1256 of the full-length DIG-3protein). As exemplified, after elimination of the deletion sequence, aninitiator methionine codon is added to the beginning of the remainingcoding sequence to restore a functional open reading frame. Also asdescribed, an additional glycine codon is to be added between themethionine codon and the codon for the instability-determining aminoacid in the instance that removal of the deleted sequence leaves exposedat the N-terminus of the remaining portion of the full-length proteinone of the instability-determining amino acids as provided above.

Table 3 describes specific variants designed in accordance with thestrategy described above.

TABLE 3 Deletion variant protein sequences of the full-length DIG-3protein of SEQ ID NO: 2 and the fusion protein sequence of SEQ ID NO: 5.Residues Residues DIG-3 added at Residues DIG-3 added at ResiduesDeletion NH2 of SEQ ID Deletion NH2 of SEQ ID Variant terminus NO: 2Variant terminus NO: 5 1 M 73-1256 68 M 73-1188 2 MG 73-1256 69 MG73-1188 3 M 74-1256 70 M 74-1188 4 MG 74-1256 71 MG 74-1188 5 M 75-125672 M 75-1188 6 M 76-1256 73 M 76-1188 7 M 77-1256 74 M 77-1188 8 M78-1256 75 M 78-1188 9 MG 78-1256 76 MG 78-1188 10 M 79-1256 77 M79-1188 11 M 80-1256 78 M 80-1188 12 M 81-1256 79 M 81-1188 13 MG81-1256 80 MG 81-1188 14 M 82-1256 81 M 82-1188 15 MG 82-1256 82 MG82-1188 16 M 83-1256 83 M 83-1188 17 M 84-1256 84 M 84-1188 18 MG84-1256 85 MG 84-1188 19 M 85-1256 86 M 85-1188 20 MG 85-1256 87 MG85-1188 21 M 86-1256 88 M 86-1188 22 M 87-1256 89 M 87-1188 23 M 88-125690 M 88-1188 24 M 89-1256 91 M 89-1188 25 MG 89-1256 92 MG 89-1188 26 M90-1256 93 M 90-1188 27 MG 90-1256 94 MG 90-1188 28 M 91-1256 95 M91-1188 29 MG 91-1256 96 MG 91-1188 30 M 92-1256 97 M 92-1188 31 M93-1256 98 M 93-1188 32 M 94-1256 99 M 94-1188 33 M 95-1256 100 M95-1188 34 MG 95-1256 101 MG 95-1188 35 M 96-1256 102 M 96-1188 36 MG96-1256 103 MG 96-1188 37 M 97-1256 104 M 97-1188 38 MG 97-1256 105 MG97-1188 39 M 98-1256 106 M 98-1188 40 MG 98-1256 107 MG 98-1188 41 M99-1256 108 M 99-1188 42 MG 99-1256 109 MG 99-1188 43 M 100-1256  110 M100-1188  44 MG 100-1256  111 MG 100-1188  45 M 101-1256  112 M101-1188  46 MG 101-1256  113 MG 101-1188  47 M 102-1256  114 M102-1188  48 MG 102-1256  115 MG 102-1188  49 M 103-1256  116 M103-1188  50 MG 103-1256  117 MG 103-1188  51 M 104-1256  118 M104-1188  52 M 105-1256  119 M 105-1188  53 MG 105-1256  120 MG105-1188  54 M 106-1256  121 M 106-1188  55 MG 106-1256  122 MG106-1188  56 M 107-1256  123 M 107-1188  57 MG 107-1256  124 MG107-1188  58 M 108-1256  125 M 108-1188  59 MG 108-1256  126 MG108-1188  60 M 109-1256  127 M 109-1188  61 MG 109-1256  128 MG109-1188  62 M 110-1256  129 M 110-1188  63 MG 110-1256  130 MG110-1188  64 M 111-1256  131 M 111-1188  65 MG 111-1256  132 MG111-1188  66 M 112-1356  133 M 112-1356  67 M 113-1256  134 M 113-1188 

Nucleic acids encoding the toxins described in Table 3 are designed inaccordance with the general principles for synthetic genes intended forexpression in plants, as discussed above.

Example 3 Design of a Plant-Optimized Version of the Coding Sequence forthe DIG-3 B.t. Insecticidal Protein

A DNA sequence having a plant codon bias was designed and synthesized toproduce the DIG-3 protein in transgenic monocot and dicot plants. Acodon usage table for maize (Zea mays L.) was calculated from 706protein coding sequences (CDs) obtained from sequences deposited inGenBank. Codon usage tables for tobacco (Nicotiana tabacum, 1268 CDs),canola (Brassica napus, 530 CDs), cotton (Gossypium hirsutum, 197 CDs),and soybean (Glycine max; ca. 1000 CDs) were downloaded from data at thewebsite http://www.kazusa.or.jp/codon/. A biased codon set thatcomprises highly used codons common to both maize and dicot datasets, inappropriate weighted average relative amounts, was calculated afteromitting any redundant codon used less than about 10% of total codonuses for that amino acid in either plant type. To derive a plantoptimized sequence encoding the DIG-3 protein, codon substitutions tothe experimentally determined DIG-3 DNA sequence were made such that theresulting DNA sequence had the overall codon composition of theplant-optimized codon bias table. Further refinements of the sequencewere made to eliminate undesirable restriction enzyme recognition sites,potential plant intron splice sites, long runs of A/T or C/G residues,and other motifs that might interfere with RNA stability, transcription,or translation of the coding region in plant cells. Other changes weremade to introduce desired restriction enzyme recognition sites, and toeliminate long internal Open Reading Frames (frames other than +1).These changes were all made within the constraints of retaining theplant-biased codon composition. Synthesis of the designed sequence wasperformed by a commercial vendor (DNA2.0, Menlo Park, Calif.).

Additional guidance regarding the production of synthetic genes can befound in, for example, WO 97/13402 and U.S. Pat. No. 5,380,831.

A plant-optimized DNA sequence encoding the full length DIG-3 toxin isgiven in SEQ ID NO:3. A dicot-optimized DNA sequence encoding the Cry1Abprotoxin segment is disclosed as SEQ ID NO:6. A maize-optimized DNAsequence encoding the Cry1Ab protoxin segment is disclosed as SEQ IDNO:7.

Example 4 Construction of Expression Plasmids Encoding DIG-3Insecticidal Toxin and Expression in Bacterial Hosts

Standard cloning methods were used in the construction of Pseudomonasfluorescens (Pf) expression plasmids engineered to produce full-lengthDIG-3 proteins encoded by plant-optimized coding regions. Restrictionendonucleases were obtained from New England BioLabs (NEB; Ipswich,Mass.) and T4 DNA Ligase (Invitrogen) was used for DNA ligation. Plasmidpreparations were performed using the NucleoBond® Xtra Kit(Macherey-Nagel Inc, Bethlehem, Pa.) or the Plasmid Midi Kit® (Qiagen),following the instructions of the suppliers. DNA fragments were purifiedusing the Millipore Ultrafree®-DA cartridge (Billerica, Mass.) afteragarose Tris-acetate gel electrophoresis.

The basic cloning strategy entailed subcloning the DIG-3 toxin codingsequence (CDS) into pDOW1169 at the SpeI and XhoI restriction sites,whereby it is placed under the expression control of the Ptac promoterand the rrnBT1T2 terminator from plasmid pKK223-3 (PL Pharmacia,Milwaukee, Wis.). pDOW1169 is a medium copy plasmid with the RSF1010origin of replication, a pyrF gene, and a ribosome binding sitepreceding the restriction enzyme recognition sites into which DNAfragments containing protein coding regions may be introduced, (USApplication 20080193974). The expression plasmid, designated pDAB4171,was transformed by electroporation into DC454 (a near wild-type P.fluorescens strain having mutations ΔpyrF and lsc::lacI^(QI)), or itsderivatives, recovered in SOC-Soy hydrolysate medium, and plated onselective medium (M9 glucose agar lacking uracil, Sambrook et al.,supra). Details of the microbiological manipulations are available inSquires et al., (2004), US Patent Application 20060008877, US PatentApplication 20080193974, and US Patent Application 20080058262,incorporated herein by reference. Colonies were first screened by PCRand positive clones were then analyzed by restriction digestion ofminiprep plasmid DNA. Plasmid DNA of selected clones containing insertswas sequenced, either by using Big Dye® Terminator version 3.1 asrecommended by the suppler (Applied Biosystems/Invitrogen), or bycontract with a commercial sequencing vendor (MWG Biotech, Huntsville,Ala.). Sequence data were assembled and analyzed using the Sequencher™software (Gene Codes Corp., Ann Arbor, Mich.).

Growth and Expression Analysis in Shake Flasks. Production of DIG-3toxin for characterization and insect bioassay was accomplished byshake-flask-grown P. fluorescens strains harboring expression constructs(e.g. clone DP2826). Seed cultures grown in M9 medium supplemented with1% glucose and trace elements were used to inoculate 50 mL of definedminimal medium with 5% glycerol (Teknova Catalog No. 3D7426, Hollister,Calif.). Expression of the DIG-3 toxin gene via the Ptac promoter wasinduced by addition of isopropyl-β-D-1-thiogalactopyranoside (IPTG)after an initial incubation of 24 hours at 30° with shaking. Cultureswere sampled at the time of induction and at various timespost-induction. Cell density was measured by optical density at 600 nm(OD₆₀₀). Other culture media suitable for growth of Pseudomonasfluorescens may also be utilized, for example, as described in Huang etal. (2007) and US Patent Application 20060008877.

Cell Fractionation and SDS-PAGE Analysis of Shake Flask Samples. At eachsampling time, the cell density of samples was adjusted to OD₆₀₀=20 and1 mL aliquots were centrifuged at 14000×g for five minutes. The cellpellets were frozen at −80°. Soluble and insoluble fractions from frozenshake flask cell pellet samples were generated using EasyLyse™ BacterialProtein Extraction Solution (EPICENTRE® Biotechnologies, Madison, Wis.).Each cell pellet was resuspended in 1 mL EasyLyse™ solution and furtherdiluted 1:4 in lysis buffer and incubated with shaking at roomtemperature for 30 minutes. The lysate was centrifuged at 14,000 rpm for20 minutes at 4° and the supernatant was recovered as the solublefraction. The pellet (insoluble fraction) was then resuspended in anequal volume of phosphate buffered saline (PBS; 11.9 mM Na₂HPO₄, 137 mMNaCl, 2.7 mM KCl, pH7.4).

Samples were mixed 1:1 with 2× Laemmli sample buffer containingβ-mercaptoethanol (Sambrook et al., supra.) and boiled for 5 minutesprior to loading onto Criterion XT® Bis-Tris 12% gels (Bio-Rad Inc.,Hercules, Calif.) Electrophoresis was performed in the recommended XTMOPS buffer. Gels were stained with Bio-Safe Coomassie Stain accordingto the manufacturer's (Bio-Rad) protocol and imaged using the AlphaInnotech Imaging system (San Leandro, Calif.).

Inclusion body preparation. Cry protein inclusion body (IB) preparationswere performed on cells from P. fluorescens fermentations that producedinsoluble B.t. insecticidal protein, as demonstrated by SDS-PAGE andMALDI-MS (Matrix Assisted Laser Desorption/Ionization MassSpectrometry). P. fluorescens fermentation pellets were thawed in a 37°water bath. The cells were resuspended to 25% w/v in lysis buffer (50 mMTris, pH 7.5, 200 mM NaCl, 20 mM EDTA disodium salt(Ethylenediaminetetraacetic acid), 1% Triton X-100, and 5 mMDithiothreitol (DTT); 5 mL/L of bacterial protease inhibitor cocktail(P8465 Sigma-Aldrich, St. Louis, Mo.) were added just prior to use). Thecells were suspended using a hand-held homogenizer at lowest setting(Tissue Tearor, BioSpec Products, Inc., Bartlesville, Okla.). Lysozyme(25 mg of Sigma L7651, from chicken egg white) was added to the cellsuspension by mixing with a metal spatula, and the suspension wasincubated at room temperature for one hour. The suspension was cooled onice for 15 minutes, then sonicated using a Branson Sonifier 250 (two1-minute sessions, at 50% duty cycle, 30% output). Cell lysis waschecked by microscopy. An additional 25 mg of lysozyme were added ifnecessary, and the incubation and sonication were repeated. When celllysis was confirmed via microscopy, the lysate was centrifuged at11,500×g for 25 minutes (4°) to form the IB pellet, and the supernatantwas discarded. The IB pellet was resuspended with 100 mL lysis buffer,homogenized with the hand-held mixer and centrifuged as above. The IBpellet was repeatedly washed by resuspension (in 50 mL lysis buffer),homogenization, sonication, and centrifugation until the supernatantbecame colorless and the IB pellet became firm and off-white in color.For the final wash, the IB pellet was resuspended in sterile-filtered(0.22 μm) distilled water containing 2 mM EDTA, and centrifuged. Thefinal pellet was resuspended in sterile-filtered distilled watercontaining 2 mM EDTA, and stored in 1 mL aliquots at −80°.

SDS-PAGE analysis and quantitation of protein in IB preparations wasdone by thawing a 1 mL aliquot of IB pellet and diluting 1:20 withsterile-filtered distilled water. The diluted sample was then boiledwith 4× reducing sample buffer [250 mM Tris, pH6.8, 40% glycerol (v/v),0.4% Bromophenol Blue (w/v), 8% SDS (w/v) and 8% 13-Mercapto-ethanol(v/v)] and loaded onto a Novex® 4-20% Tris-Glycine, 12+2 well gel(Invitrogen) run with 1× Tris/Glycine/SDS buffer (BioRad). The gel wasrun for 60 min at 200 volts then stained with Coomassie Blue (50%G-250/50% R-250 in 45% methanol, 10% acetic acid), and destained with 7%acetic acid, 5% methanol in distilled water. Quantification of targetbands was done by comparing densitometric values for the bands againstBovine Serum Albumin (BSA) samples run on the same gel to generate astandard curve.

Solubilization of Inclusion Bodies. Six mL of inclusion body suspensionfrom Pf clone DP2826 (containing 32 mg/mL of DIG-3 protein) werecentrifuged on the highest setting of an Eppendorf model 5415C microfuge(approximately 14,000×g) to pellet the inclusions. The storage buffersupernatant was removed and replaced with 25 mL of 100 mM sodiumcarbonate buffer, pH11, in a 50 mL conical tube. Inclusions wereresuspended using a pipette and vortexed to mix thoroughly. The tube wasplaced on a gently rocking platform at 4° overnight to extract thetarget protein. The extract was centrifuged at 30,000×g for 30 min at4°, and the resulting supernatant was concentrated 5-fold using anAmicon Ultra-15 regenerated cellulose centrifugal filter device (30,000Molecular Weight Cutoff; Millipore). The sample buffer was then changedto 10 mM CAPS [3-(cyclohexamino)1-propanesulfonic acid] pH 10, usingdisposable PD-10 columns (GE Healthcare, Piscataway, N.J.).

Gel electrophoresis. The concentrated extract was prepared forelectrophoresis by diluting 1:50 in NuPAGE® LDS sample buffer(Invitrogen) containing 5 mM dithiothreitol as a reducing agent andheated at 95° for 4 minutes. The sample was loaded in duplicate lanes ofa 4-12% NuPAGE® gel alongside five BSA standards ranging from 0.2 to 2μg/lane (for standard curve generation). Voltage was applied at 200Vusing MOPS SDS running buffer (Invitrogen) until the tracking dyereached the bottom of the gel. The gel was stained with 0.2% CoomassieBlue G-250 in 45% methanol, 10% acetic acid, and destained, firstbriefly with 45% methanol, 10% acetic acid, and then at length with 7%acetic acid, 5% methanol until the background cleared. Followingdestaining, the gel was scanned with a Biorad Fluor-S MultiImager. Theinstrument's Quantity One v.4.5.2 Software was used to obtainbackground-subtracted volumes of the stained protein bands and togenerate the BSA standard curve that was used to calculate theconcentration of DIG-3 protein in the stock solution.

Example 5 Insecticidal Activity of Modified DIG-3 Protein Produced inPseudomonas fluorescens

DIG-3 B.t. insecticidal toxin was demonstrated to be active onLepidopteran species including the European corn borer (ECB; Ostrinianubilalis (Hübner)), cry1F-resistant ECB (rECB), diamondback moth (DBM;Plutella xylostella (Linnaeus)), cry1A-resistant DBM (rDBM), cornearworm (CEW; Helicoverpa zea (Boddie)), black cutworm (BCW; Agrotisipsilon (Hufnagel)), tobacco budworm (TBW; Heliothis virescens(Fabricius)), and cabbage looper (CL; Trichoplusia ni (Hübner)). DIG-3protein was also tested for activity on fall armyworm (FAW, Spodopterafrugiperda), Cry1F-resistant FAW (rFAW) and western corn rootworm (WCR,Diabrotica virgifera virgifera LeConte).

Sample preparation and bioassays. Inclusion body preparations in 10 mMCAPS pH10 were diluted appropriately in 10 mM CAPS pH 10, and allbioassays contained a control treatment consisting of this buffer, whichserved as a background check for mortality or growth inhibition.

Protein concentrations in bioassay buffer were estimated by gelelectrophoresis using BSA to create a standard curve for geldensitometry, which was measured using a BioRad imaging system (Fluor-SMultiImager with Quantity One software version 4.5.2). Proteins in thegel matrix were stained with Coomassie Blue-based stain and destainedbefore reading.

Purified proteins were tested for insecticidal activity in bioassaysconducted with neonate Lepidopteran larvae on artificial insect diet.Larvae of BCW, CEW, CL, DBM, rDBM, ECB, FAW and TBW were hatched fromeggs obtained from a colony maintained by a commercial insectary (BenzonResearch Inc., Carlisle, Pa.). WCR eggs were obtained from CropCharacteristics, Inc. (Farmington, Minn.). Larvae of rECB and rFAW werehatched from eggs harvested from proprietary colonies (Dow AgroSciencesLLC, Indianapolis, Ind.).

The bioassays were conducted in 128-well plastic trays specificallydesigned for insect bioassays (C-D International, Pitman, N.J.). Eachwell contained 1.0 mL of Multi-species Lepidoptera diet (SouthlandProducts, Lake Village, Ariz.). A 40 μL aliquot of protein sample wasdelivered by pipette onto the 1.5 cm² diet surface of each well (26.7μL/cm²). Diet concentrations were calculated as the amount (ng) of DIG-3protein per square centimeter (cm²) of surface area in the well. Thetreated trays were held in a fume hood until the liquid on the dietsurface had evaporated or was absorbed into the diet.

Within a few hours of eclosion, individual larvae were picked up with amoistened camel hair brush and deposited on the treated diet, one larvaper well. The infested wells were then sealed with adhesive sheets ofclear plastic, vented to allow gas exchange (C-D International, Pitman,N.J.). Bioassay trays were held under controlled environmentalconditions (28° C., ˜40% Relative Humidity, 16:8 [Light:Dark]) for 5days, after which the total number of insects exposed to each proteinsample, the number of dead insects, and the weight of surviving insectswere recorded. Percent mortality and percent growth inhibition werecalculated for each treatment. Growth inhibition (GI) was calculated asfollows:

GI=[1−(TWIT/TNIT)/(TWIBC/TNIBC)]

where TWIT is the Total Weight of Insects in the Treatment,

TNIT is the Total Number of Insects in the Treatment

TWIBC is the Total Weight of Insects in the Background Check (Buffercontrol), and

TNIBC is the Total Number of Insects in the Background Check (Buffercontrol).

The GI₅₀ was determined to be the concentration of DIG-3 protein in thediet at which the GI value was 50%. The LC₅₀ (50% Lethal Concentration)was recorded as the concentration of DIG-3 protein in the diet at which50% of test insects were killed. Statistical analysis (One-way ANOVA)was done using JMP software (SAS, Cary, N.C.)

Table 6 presents the results of bioassay tests of DIG-3 protein onEuropean corn borer and cry1F-resistant European corn borer (rECB). Anunexpected and surprising finding is that the rECB test insects were assusceptible to the action of DIG-3 protein as were the wild type ECBinsects.

TABLE 6 LC₅₀ and GI₅₀ values calculated for ECB and rECB, withConfidence Intervals (CI) of 95% Insect LC₅₀ (ng/cm²) 95% CI GI₅₀(ng/cm²) 95% CI ECB 591.9 308.1-1315.3 122.6 45.6-328.4  rECB 953.6534.1-1953.6 270.9 53.0-1382.2

Table 7 presents the results of bioassays on a broad spectrum ofLepidopteran and a Coleopteran pest (WCR). The DIG-3 protein hasunexpected and surprising activity on diamondback moth as well as rDBM.Further the DIG-3 Cry protein is effective in controlling the growth ofseveral other Lepidopteran insects.

TABLE 7 Insecticidal and growth inhibitory effects of DIG-3 proteiningested by test insects Test Response at Insect 9000 ng/cm² StatisticalAnalysis* DBM 100% mortality rDBM 100% mortality CL 75% mortality,significant GI (GI) P < 0.001, df = 1, α = 0.05 CEW Significant GI (GI)P = 0.02, df = 1, α = 0.05 TBW Visible GI, some mortality Not availableBCW Visible GI Not available FAW No activity observed rFAW No activityobserved WCR No activity observed *GI = Growth Inhibition. P-value =test statistic. df = Degrees of Freedom, α(alpha) level 0.05 = the levelof test significance.

Example 6 Agrobacterium Transformation

Standard cloning methods are used in the construction of binary planttransformation and expression plasmids. Restriction endonucleases and T4DNA Ligase are obtained from NEB. Plasmid preparations are performedusing the NucleoSpin® Plasmid Preparation kit or the NucleoBond® AX XtraMidi kit (both from Macherey-Nagel), following the instructions of themanufacturers. DNA fragments are purified using the QIAquick® PCRPurification Kit or the QIAEX II® Gel Extraction Kit (both from Qiagen)after gel isolation.

DNA fragments comprising nucleotide sequences that encode the DIG-3protein in native or modified forms, or fragments thereof, may besynthesized by a commercial vendor (e.g. DNA2.0, Menlo Park, Calif.) andsupplied as cloned fragments in standard plasmid vectors, or may beobtained by standard molecular biology manipulation of other constructscontaining appropriate nucleotide sequences. Unique restriction sitesinternal to a DIG-3 coding region may be identified and DNA fragmentscomprising the sequences between the restriction sites of the DIG-3coding region may be synthesized, each such fragment encoding a specificdeletion, insertion or other DIG-3 variation. The DNA fragments encodingthe modified DIG-3 fragments may be joined to other DIG-3 coding regionfragments or other Cry coding region fragments at appropriaterestriction sites to obtain a coding region encoding the desiredfull-length DIG-3 protein, deleted or variant DIG-3 protein, or fusedprotein. For example, one may identify an appropriate restrictionrecognition site at the start of a first DIG-3 coding region, and asecond restriction site internal to the DIG-3 coding region. Cleavage ofthis first DIG-3 coding region at these restriction sites would generatea DNA fragment comprising part of the first DIG-3 coding region. Asecond DNA fragment flanked by analogously-situated compatiblerestriction sites specific for another DIG-3 coding region or other Crycoding region may be used in combination with the first DNA restrictionfragment to construct a variant or fused clone.

In a non-limiting example, a basic cloning strategy may be to subclonefull length or modified DIG-3 coding sequences (CDS) into a plantexpression plasmid at NcoI and Sad restriction sites. The resultingplant expression cassettes containing the appropriate DIG-3 codingregion under the control of plant expression elements, (e.g., plantexpressible promoters, 3′ terminal transcription termination andpolyadenylate addition determinants, and the like) are subcloned into abinary vector plasmid, utilizing, for example, Gateway® technology orstandard restriction enzyme fragment cloning procedures. LR Clonase™(Invitrogen) for example, may be used to recombine the full length andmodified gene plant expression cassettes into a binary planttransformation plasmid if the Gateway® technology is utilized. It isconvenient to employ a binary plant transformation vector that harbors abacterial gene that confers resistance to the antibiotic spectinomycinwhen the plasmid is present in E. coli and Agrobacterium cells. It isalso convenient to employ a binary vector plasmid that contains aplant-expressible selectable marker gene that is functional in thedesired host plants. Examples of plant-expressible selectable markergenes include but are not limited those that encode the aminoglycosidephosphotransferase gene (aphII) of transposon Tn5, which confersresistance to the antibiotics kanamycin, neomycin and G418, as well asthose genes which code for resistance or tolerance to glyphosate;hygromycin; methotrexate; phosphinothricin (bialaphos), imidazolinones,sulfonylureas and triazolopyrimidine herbicides, such as chlorosulfuron,bromoxynil, dalapon and the like.

Electro-competent cells of Agrobacterium tumefaciens strain Z707S (astreptomycin-resistant derivative of Z707; Hepburn et al., 1985) areprepared and transformed using electroporation (Weigel and Glazebrook,2002). After electroporation, 1 mL of YEP broth (gm/L: yeast extract,10; peptone, 10; NaCl, 5) are added to the cuvette and the cell-YEPsuspension is transferred to a 15 mL culture tube for incubation at 28°in a water bath with constant agitation for 4 hours. The cells areplated on YEP plus agar (25 gm/L) with spectinomycin (200 μg/mL) andstreptomycin (250 μg/mL) and the plates are incubated for 2-4 days at28°. Well separated single colonies are selected and streaked onto freshYEP+agar plates with spectinomycin and streptomycin as before, andincubated at 28° for 1-3 days.

The presence of the DIG-3 gene insert in the binary plant transformationvector is performed by PCR analysis using vector-specific primers withtemplate plasmid DNA prepared from selected Agrobacterium colonies. Thecell pellet from a 4 mL aliquot of a 15 mL overnight culture grown inYEP with spectinomycin and streptomycin as before is extracted usingQiagen Spin® Mini Preps, performed per manufacturer's instructions.Plasmid DNA from the binary vector used in the Agrobacteriumelectroporation transformation is included as a control. The PCRreaction is completed using Taq DNA polymerase from Invitrogen permanufacture's instructions at 0.5× concentrations. PCR reactions arecarried out in a MJ Research Peltier Thermal Cycler programmed with thefollowing conditions: Step 1) 94° for 3 minutes; Step 2) 94° for 45seconds; Step 3) 55° for 30 seconds; Step 4) 72° for 1 minute per kb ofexpected product length; Step 5) 29 times to Step 2; Step 6) 72° for 10minutes. The reaction is maintained at 4° after cycling. Theamplification products are analyzed by agarose gel electrophoresis (e.g.0.7% to 1% agarose, w/v) and visualized by ethidium bromide staining. Acolony is selected whose PCR product is identical to the plasmidcontrol.

Alternatively, the plasmid structure of the binary plant transformationvector containing the DIG-3 gene insert is performed by restrictiondigest fingerprint mapping of plasmid DNA prepared from candidateAgrobacterium isolates by standard molecular biology methods well knownto those skilled in the art of Agrobacterium manipulation.

Those skilled in the art of obtaining transformed plants viaAgrobacterium-mediated transformation methods will understand that otherAgrobacterium strains besides Z7075 may be used to advantage, and thechoice of strain may depend upon the identity of the host plant speciesto be transformed.

Example 7

Production of DIG-3 B.t. Insecticidal Proteins and Variants in DicotPlants

Arabidopsis Transformation. Arabidopsis thaliana Col-01 is transformedusing the floral dip method (Weigel and Glazebrook, 2002). The selectedAgrobacterium colony is used to inoculate 1 mL to 15 mL cultures of YEPbroth containing appropriate antibiotics for selection. The culture isincubated overnight at 28° with constant agitation at 220 rpm. Eachculture is used to inoculate two 500 mL cultures of YEP broth containingappropriate antibiotics for selection and the new cultures are incubatedovernight at 28° with constant agitation. The cells are pelleted atapproximately 8700×g for 10 minutes at room temperature, and theresulting supernatant is discarded. The cell pellet is gentlyresuspended in 500 mL of infiltration media containing: ½× Murashige andSkoog salts (Sigma-Aldrich)/Gamborg's B5 vitamins (Gold BioTechnology,St. Louis, Mo.), 10% (w/v) sucrose, 0.044 μM benzylaminopurine (10μL/liter of 1 mg/mL stock in DMSO) and 300 μL/liter Silwet L-77. Plantsapproximately 1 month old are dipped into the media for 15 seconds, withcare taken to assure submergence of the newest inflorescence. The plantsare then laid on their sides and covered (transparent or opaque) for 24hours, washed with water, and placed upright. The plants are grown at22°, with a 16-hour light/8-hour dark photoperiod. Approximately 4 weeksafter dipping, the seeds are harvested.

Arabidopsis Growth and Selection. Freshly harvested T1 seed is allowedto dry for at least 7 days at room temperature in the presence ofdesiccant. Seed is suspended in a 0.1% agar/water (Sigma-Aldrich)solution and then stratified at 4° for 2 days. To prepare for planting,Sunshine Mix LP5 (Sun Gro Horticulture Inc., Bellevue, Wash.) in 10.5inch×21 inch germination trays (T.O. Plastics Inc., Clearwater, Minn.)is covered with fine vermiculite, sub-irrigated with Hoagland's solution(Hoagland and Arnon, 1950) until wet, then allowed to drain for 24hours. Stratified seed is sown onto the vermiculite and covered withhumidity domes (KORD Products, Bramalea, Ontario, Canada) for 7 days.Seeds are germinated and plants are grown in a Conviron (Models CMP4030or CMP3244; Controlled Environments Limited, Winnipeg, Manitoba, Canada)under long day conditions (16 hours light/8 hours dark) at a lightintensity of 120-150 μmmol/m² sec under constant temperature (22°) andhumidity (40-50%). Plants are initially watered with Hoagland's solutionand subsequently with deionized water to keep the soil moist but notwet.

The domes are removed 5-6 days post sowing and plants are sprayed with achemical selection agent to kill plants germinated from nontransformedseeds. For example, if the plant expressible selectable marker geneprovided by the binary plant transformation vector is a pat or bar gene(Wehrmann et al., 1996), transformed plants may be selected by sprayingwith a 1000× solution of Finale (5.78% glufosinate ammonium, FarnamCompanies Inc., Phoenix, Ariz.). Two subsequent sprays are performed at5-7 day intervals. Survivors (plants actively growing) are identified7-10 days after the final spraying and transplanted into pots preparedwith Sunshine Mix LP5. Transplanted plants are covered with a humiditydome for 3-4 days and placed in a Conviron under the above-mentionedgrowth conditions.

Those skilled in the art of dicot plant transformation will understandthat other methods of selection of transformed plants are available whenother plant expressible selectable marker genes (e.g. herbicidetolerance genes) are used.

Insect Bioassays of transgenic Arabidopsis. Transgenic Arabidopsis linesexpressing modified Cry proteins are demonstrated to be active againstsensitive insect species in artificial diet overlay assays. Proteinextracted from transgenic and non-transgenic Arabidopsis lines isquantified by appropriate methods and sample volumes are adjusted tonormalize protein concentration. Bioassays are conducted on artificialdiet as described above. Non-transgenic Arabidopsis and/or buffer andwater are included in assays as background check treatments.

Example 8 Agrobacterium Transformation for Generation of SuperbinaryVectors

The Agrobacterium superbinary system is conveniently used fortransformation of monocot plant hosts. Methodologies for constructingand validating superbinary vectors are well disclosed and incorporatedherein by reference (Operating Manual for Plasmid pSB1, Version 3.1,available from Japan Tobacco, Inc., Tokyo, Japan). Standard molecularbiological and microbiological methods are used to generate superbinaryplasmids. Verification/validation of the structure of the superbinaryplasmid is done using methodologies as described above for binaryvectors, and may be modified as suggested in the Operating Manual forPlasmid pSB1.

Example 9 Production of DIG-3 B.t. Insecticidal Proteins and Variants inMonocot Plants

Agrobacterium-Mediated Transformation of Maize. Seeds from a High II F₁cross (Armstrong et al., 1991) are planted into 5-gallon-pots containinga mixture of 95% Metro-Mix 360 soilless growing medium (Sun GroHorticulture, Bellevue, Wash.) and 5% clay/loam soil. The plants aregrown in a greenhouse using a combination of high pressure sodium andmetal halide lamps with a 16:8 hour Light:Dark photoperiod. Forobtaining immature F₂ embryos for transformation, controlledsib-pollinations are performed. Immature embryos are isolated at 8-10days post-pollination when embryos are approximately 1.0 to 2.0 mm insize.

Infection and co-cultivation. Maize ears are surface sterilized byscrubbing with liquid soap, immersing in 70% ethanol for 2 minutes, andthen immersing in 20% commercial bleach (0.1% sodium hypochlorite) for30 minutes before being rinsed with sterile water. A suspension ofAgrobacterium cells containing a superbinary vector is prepared bytransferring 1-2 loops of bacteria grown on YEP solid medium containing100 mg/L spectinomycin, 10 mg/L tetracycline, and 250 mg/L streptomycinat 28° for 2-3 days into 5 mL of liquid infection medium (LS BasalMedium (Linsmaier and Skoog, 1965), N6 vitamins (Chu et al., 1975), 1.5mg/L 2,4-Dichlorophenoxyacetic acid (2,4-D), 68.5 gm/L sucrose, 36.0gm/L glucose, 6 mM L-proline, pH 5.2) containing 100 μM acetosyringone.The solution was vortexed until a uniform suspension was achieved, andthe concentration is adjusted to a final density of about 200 Klettunits, using a Klett-Summerson colorimeter with a purple filter, or anoptical density of approximately 0.4 at 550 nm. Immature embryos areisolated directly into a micro centrifuge tube containing 2 mL of theinfection medium. The medium is removed and replaced with 1 mL of theAgrobacterium solution with a density of 200 Klett units, and theAgrobacterium and embryo solution is incubated for 5 minutes at roomtemperature and then transferred to co-cultivation medium (LS BasalMedium, N6 vitamins, 1.5 mg/L 2,4-D, 30.0 gm/L sucrose, 6 mM L-proline,0.85 mg/L AgNO₃, 100 μM acetosyringone, 3.0 gm/L Gellan gum(PhytoTechnology Laboratories., Lenexa, Kans.), pH 5.8) for 5 days at25° under dark conditions.

After co-cultivation, the embryos are transferred to selective mediumafter which transformed isolates are obtained over the course ofapproximately 8 weeks. For selection of maize tissues transformed with asuperbinary plasmid containing a plant expressible pat or bar selectablemarker gene, an LS based medium (LS Basal medium, N6 vitamins, 1.5 mg/L2,4-D, 0.5 gm/L MES (2-(N-morpholino)ethanesulfonic acid monohydrate;PhytoTechnologies Labr.), 30.0 gm/L sucrose, 6 mM L-proline, 1.0 mg/LAgNO₃, 250 mg/L cefotaxime, 2.5 gm/L Gellan gum, pH 5.7) is used withBialaphos (Gold BioTechnology). The embryos are transferred to selectionmedia containing 3 mg/L Bialaphos until embryogenic isolates wereobtained. Recovered isolates are bulked up by transferring to freshselection medium at 2-week intervals for regeneration and furtheranalysis.

Those skilled in the art of maize transformation will understand thatother methods of selection of transformed plants are available whenother plant expressible selectable marker genes (e.g. herbicidetolerance genes) are used.

Regeneration and seed production. For regeneration, the cultures aretransferred to “28” induction medium (MS salts and vitamins, 30 gm/Lsucrose, 5 mg/L Benzylaminopurine, 0.25 mg/L 2,4-D, 3 mg/L Bialaphos,250 mg/L cefotaxime, 2.5 gm/L Gellan gum, pH 5.7) for 1 week underlow-light conditions (14 μEm⁻²s⁻¹) then 1 week under high-lightconditions (approximately 89 μEm⁻²s⁻¹). Tissues are subsequentlytransferred to “36” regeneration medium (same as induction medium exceptlacking plant growth regulators). When plantlets grow to 3-5 cm inlength, they were transferred to glass culture tubes containing SHGAmedium (Schenk and Hildebrandt salts and vitamins (1972);PhytoTechnologies Labr.), 1.0 gm/L myo-inositol, 10 gm/L sucrose and 2.0gm/L Gellan gum, pH 5.8) to allow for further growth and development ofthe shoot and roots. Plants are transplanted to the same soil mixture asdescribed earlier herein and grown to flowering in the greenhouse.Controlled pollinations for seed production are conducted.

Example 10 Bioassay of Transgenic Maize

Bioactivity of the DIG-3 protein and variants produced in plant cells isdemonstrated by conventional bioassay methods (see, for example Huang etal., 2006). One is able to demonstrate efficacy, for example, by feedingvarious plant tissues or tissue pieces derived from a plant producing aDIG-3 toxin to target insects in a controlled feeding environment.Alternatively, protein extracts may be prepared from various planttissues derived from a plant producing the DIG-3 toxin and incorporatethe extracted proteins in an artificial diet bioassay as previouslydescribed herein. It is to be understood that the results of suchfeeding assays are to be compared to similarly conducted bioassays thatemploy appropriate control tissues from host plants that do not producethe DIG-3 protein or variants, or to other control samples.

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1. An isolated polypeptide comprising a core toxin segment selected fromthe group consisting of (a) a polypeptide comprising the amino acidsequence of residues 113 to 643 of SEQ ID NO: 2; (b) a polypeptidecomprising an amino acid sequence having at least 90% sequence identityto the amino acid sequence of residues 113 to 643 of SEQ ID NO:2; (c) apolypeptide comprising an amino acid sequence of residues 113 to 643 ofSEQ ID NO: 2 with up to 20 amino acid substitutions, deletions, ormodifications that do not adversely affect expression or activity of thetoxin encoded by SEQ ID NO: 2; or an insecticidally active fragmentthereof.
 2. The isolated polypeptide of claim 1 comprising a core toxinsegment selected from the group consisting of (a) a polypeptidecomprising the amino acid sequence of residues 73 to 643 of SEQ ID NO:2; (b) a polypeptide comprising an amino acid sequence having at least90% sequence identity to the amino acid sequence of residues 73 to 643of SEQ ID NO:2; (c) a polypeptide comprising an amino acid sequence ofresidues 73 to 643 of SEQ ID NO: 2 with up to 20 amino acidsubstitutions, deletions, or modifications that do not adversely affectexpression or activity of the toxin encoded by SEQ ID NO: 2; or aninsecticidally active fragment thereof.
 3. The isolated polypeptide ofclaim 1 comprising a core toxin segment selected from the groupconsisting of (a) a polypeptide comprising the amino acid sequence ofresidues 1 to 643 of SEQ ID NO: 2; (b) a polypeptide comprising an aminoacid sequence having at least 90% sequence identity to the amino acidsequence of residues 1 to 643 of SEQ ID NO:2; (c) a polypeptidecomprising an amino acid sequence of residues 1 to 643 of SEQ ID NO: 2with up to 20 amino acid substitutions, deletions, or modifications thatdo not adversely affect expression or activity of the toxin encoded bySEQ ID NO: 2; or an insecticidally active fragment thereof.
 4. A plantcomprising the polypeptide of claim
 1. 5. A method for controlling apest population comprising contacting said population with apesticidally effective amount of the polypeptide of claim
 1. 7. Anisolated nucleic acid that encodes a polypeptide of claim
 1. 8. Theisolated nucleic acid of claim 7 of SEQ ID NO: 1 or SEQ ID NO:3.
 9. Apolypeptide of claim 1 of SEQ ID NO: 2 or SEQ ID NO:5.
 10. A DNAconstruct comprising the nucleotide sequence of claim 1 operably linkedto a promoter that is not derived from Bacillus thuringiensis and iscapable of driving expression in a plant.
 11. A transgenic plant thatcomprises the DNA construct of claim 10 stably incorporated into itsgenome.
 12. A method for protecting a plant from a pest comprisingintroducing into said plant the construct of claim
 10. 13. Thepolypeptide of claim 1 having activity against European corn borer.